Metagenomes

Abstract



Importance


Introduction
Historically, b-lactam antibiotics are the most widely used antibiotics in clinical settings around the world (Garau 2005, Boeckel 2014 ESAC 2009, ECDC 2012), but the successful of antibiotic treatments is compromised by the development of resistance in many important clinical pathogens (Davies 2010). The major cause of resistance to b-lactams antibiotics are bacterial enzymes called b-lactamases with the capacity to hydrolyze the molecular structure of the b-lactam antibiotic (Davies 1994). Many studies have assessed the presence and diversity of b-lactamases in clinical settings (Paterson 2005, Rice 2001, Ramphal 2006, Shahid 2014, Sullivan 2015), but no enough efforts have been put in assess b-lactamases in non-clinical settings.

Studies assessing the AR phenomenon in non-clinical environments usually are based on functional metagenomics studies, PCR reactions, MIC tests (Donato 2010, Bhullar 2012, Segawa 2012Amos 2014, Forsberg 2014, Su 2014Ma 2014), but in the last years, the use of metagenomic approaches (Yang 2013,Li 2015Fondi 2016) appear such as an interesting tool to assess ARGs in environmental samples. In this context, the focus on antibiotic resistance genes in natural environments should be considered, specially if we consider the evidence that these genes have a long evolutionary history in natural environments (Aminov 2009, Hall 2004, Garau 2005). Thus, natural environments appear such as a reservoir of potential ARGs to pathogenic bacteria (Berglund 2015, Versluis 2015) that cannot be ignored.

Despite that in the last years more studies are focused on ARGs in the environment (Czekalski 2015, Bhullar 2012, Cristóbal-Azkarate 2014, Nesme 2014, Karkman 2016), the lack of studies focused on the high diversity of b-lactamase genes in different environments, avoid the understanding of important process with clinical implications such as b-lactamase gene transfer between environments and the impact of anthropogenic forces on both b-lactamase content and diversity in natural environments.

In this study we assess the presence and diversity of b-lacatamases in different environments through a wide metagenomic approach and network oriented analysis, providing important findings on the pool of b-lactamase genes in different environments. 


Results

An effort to understand the scope of b-lactam resistance in the environment was performed through a global survey of b-lactamases present in 232 shotgun metagenomes. Metagenomic data sets were obtained from two important public repositories. These data sets  account for 770 Gigabytes of information and more than 4.7 billion of metagenomic reads (details can be found in table S1); and are related to different environments such as agricultural soils, non-agricultural soils, glaciers, fresh water, oceans, human gut, cow feces, cow rumen and wastewater treatment plant environment. The metagenomic reads were compared to the EX-B database; a database that include more than 1500 b-lactamase genes (for details see material and methods). Metagenomic reads containing b-lactamases were identified by BLASTX  based on their similarity to the EX-B database at the cutoff thresholds described in material and methods (percent of identity equal o higher than 50%, e-value equal or lower than \(10^{-5}\), and a bit score equal o higher than 30). Environmental b-lactamases can be diverse relative to clinically characterized b-lactamases (Forsberg 2014), due to we chose a percent of identity equal o higher than 50%. Reads matching b-lactamases were used to construct distance matrices and b-lactamase gene networks, which were used for downstream a