b-lactamase hits obtained by BLAST were used to construct metagenome distance matrices and b-lactamase gene networks. When all the b-lactamase hits were used to construct the gene network (Fig. S1), different cluster were clearly observable; the same trend was obtained when genes poorly described at gene level (i.e. identified only such b-latamase, ESBL gene or class A/B/C/D) were removed from the analysis (Figure 3). The network analysis indicate that each clusters harbor metagenomes almost exclusively related to a given environment; thus five cluster representing soils (including a differentiation between agricultural and non-agricultural soils), human gut, animal gut and wastewater were identified. Cluster analysis performed on b-lactamase gene networks, indicated that samples from the same environment are more tightly connected than samples from different environments (Table S2). This is clearly visible in Fig. 3, where nodes from a given environment (soils, human gut, cow gut and wastewater treatment plant environment) show more connections between them than the number of connections with nodes related to other environments.

In order to test if sample geography influences on b-lactamase gene content, nodes present in Fig. 3 were presented according their geographic origin (Fig. 4). Clearly, the results obtained here indicated that geography is not correlated with both b-lactamase gene content and diversity.

The BLAST analysis performed on metagenomic reads indicate the presence of b-lactamase genes in different environments, but also it can  indicates how close are the metagenomic reads related to the reference database. To elucidate it, we choose four clinically important b-lactamases such as blaCTX-M, blaGES, blaOXA and blaTEM and the hits obtained by BLAST were grouped in five groups  according the  percent of identity to the same genes  in five groupes X-B database  (identities from 50 to 59, 60 to 69, 70 to 79, 80 to 89 and 90 to 100 percent) (Fig. 5). The results indicate indicated  that in each case, most of the hits show a low percent of identity; thus, for blaOXA gene, 40% in average, between 43.7 (in human gut metagenomes) to 69.9% (bovine feces)  of the blaOXA  hitsor more  show a percent of identity between 50 to 59 percent. Hits with higher percent of identity to our database were obtained from human gut, wastewater treatment plant environment, fresh water and glacier metagenomes.  In the case of blaGES, 40% or more 44.2% (non-agricultural soils) of hits until 81.2 % (glaciers) of  hits show a percent of identity between 50 to 59 percent, and for percent. In addition all the obtained hits in human gut show the same range of identity, but it is based only in six hits. In the case of  blaCTX-M and blaTEM, close to 40% of the hits show a percent of identity of 50 to 59 percent. In some cases, a higher percent of identity can be observed for most of the hits in a given environment (blaCTX-M gene in human and bovine environment, environment;  blaTEM in ocean, human gut or bovine rumen) rumen metagenomes)  but in those cases, the total number of hits obtained was very low. Higher low to be taken into account. Real higher  percent of identity (between 90 to 100%) were only detected in some specific cases; cases that include a high number of hits;  thus, blaCTX-M gene shows hits with the high percent of identity in samples obtained from human gut. gut (n=14)  and  hits with high percent of identity to blaGES gene were foundonly  in wastewater treatment plant environment; in relation to blaOXA, hits with higher percent of identity environment (n=105). Hits related  toour database were obtained from human gut, wastewater treatment plant environment, fresh water and glacier metagenomes and finally,  blaTEM hits that shows high show a  percent of identity to our EX-B database were obtained from database, higher than 79%,  in  human gut, wastewater treatment plant environment, plants,  oceans, fresh water, glacier and agricultural soil metagenomes.

The metagenomes.

The  target of 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 ( ...
author = {Dennis Versluis and Marco Maria D'Andrea and Javier Ramiro Garcia and Milkha M. Leimena and Floor Hugenholtz and Jing Zhang and Ba{\c{s}}ak Öztürk and Lotta Nylund and Detmer Sipkema and Willem van Schaik and Willem M. de Vos and Michiel Kleerebezem and Hauke Smidt and Mark W.J. van Passel},  title = {Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions},  journal = {Sci. Rep.}  }" data-bib-key="Versluis_2015" contenteditable="false">Versluis 2015) that cannot be ignored.

The ignored.

The  results obtained in this study show the high content of b-lactamase genes in each analyzed environments, but a deep analysis indicate important differences in the diversity of those genes. Thus, soil and glacier metagenomes exhibit the higher diversity of b-lactamase genes (40 different b-lactamase genes in average), followed by wastewater metagenomes (32 different b-lactamase genes). The high diversity of b-lactamases in soils is in accordance with a previous metagenomic-based study of antibiotic resistance genes in the environment (