The analysis exposure. Other study, using a metagenomic approach, found that the use of antibiotics and metals cause the enrichment and release of ARGs and mobile genetic elements to the environment (Zhu 2013)

Undoubtedly,  the massive use of public  metagenome sequences, clearly indicate that projects, each of them performed by  different factor investigator and using different methodologies can  affect the b-lactamase diversity values here results  obtained. In this context is important indicate that despite that all the metagenomes share the same type of technology used in the sequencing process, differences in methodology can be expected. In the same context, despite that metagenomes can be grouped according different environments, some differences can affect the sequencing results; thus, results. For instance,  in the ocean environment, metagenomes related to the Sydney harbour metagenome project (see Table S1) were obtained at 0,3 m in depth, in the estuary of Sydney, and metagenomes related to Amazon continuum metagenomes project were obtained at depths ranged from 3.63 to 4.47 meters,and the samples were obtained  at different points, from seawater close to the cost line until seawater far distances  from the cost line (open ocean). For instance, coast. In relation to water samples,  some studies have been show the differences in the AR profile across different water layers in different lake samples ( ...
author = {Andrea Di Cesare and Ester M. Eckert and Alessia Teruggi and Diego Fontaneto and Roberto Bertoni and Cristiana Callieri and Gianluca Corno},  title = {Constitutive presence of antibiotic resistance genes within the bacterial community of a large subalpine lake},  journal = {Molecular Ecology}  }" data-bib-key="Di_Cesare_2015" contenteditable="false">Cesare 2015). In relation to the case of  soil metagenomes, the agricultural soil  metagenomes here analyzed were exposed to different agricultural practices that can affect the microbiological content on soil and therefor, affect the genomic bacterial  content on soil. Illustrating the last point,   ...
author = {Chu Thi Thanh Binh and Holger Heuer and Martin Kaupenjohann and Kornelia Smalla},  title = {Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids},  journal = {{FEMS} Microbiology Ecology}  }" data-bib-key="Binh_2008" contenteditable="false">Binh 2008 showed that manure is a hot spot of bacteria carrying antibiotic resistance genes in MGEs. Finally, in the case of gut metagenomes, clearly the health situation and/or the drug supply can affect both the microbiological and genomic content. Our analysis take into account those variability, and steps oriented to reduce it were applied. 

Supporting performed. 

Supporting  the differences in both  b-lactamase content and diversity, diversity observed across the different analyzed environments,  our indicator species analysis show habitat-specificity for some b-lactamase genes. Interestingly, Thus,  four b-lactamase genes (blaEBR, CfxA, mecA HGI) showed a high faithfulness of occurrence in the human gut environment; those environment. Those  genes have been previously identified only in humans (both health and sick) ( ...
title = {Detection of antibiotic-resistant bacteria and their resistance genes in wastewater,  surface water, and drinking water biofilms},  journal = {{FEMS} Microbiology Ecology}  }" data-bib-key="Schwartz_2003" contenteditable="false">Schwartz 2003), but there are no reports of their occurrence in natural environments. In addition, this analysis was performed when environments were categorized according degrees 

When we tried to analyzed the presence  of human impact (see results); thus, b-lactamases according anthropogenic impact,  the four previous above indicated  genesthat shown a high probability of be present in human gut metagenomes,  exhibit again the same trend, this mean show the same is a  high probability to be present in the category of higher high  anthropogenic impact. Interestingly, in the category of the lowest low  anthropogenic impact, that include glacier and non-agricultural soil metagenomes, a high number of b-lactamase genes (49) show a high faithfulness of occurrence.

On occurrence. Thus, on  the whole, our results suggest that highly anthropogenic impacted environments select for only few specific b-lactamase genes, while less impacted environments, probably with a lower level of selective pressure, may content lower levels of b-lactamases but with a high b-lactamase diversity.

Our gene network results show a high number of connections between nodes from the same type of environment, but only few connections between nodes of two different environments; suggesting that b-lactamase genes show a high intra-habitat mobility, but a limited inter-habitat transferability. Our findings are in concordance with metagenomic (Fondi et al. 2016 and ...
author = {Chris S. Smillie and Mark B. Smith and Jonathan Friedman and Otto X. Cordero and Lawrence A. David and Eric J. Alm},  title = {Ecology drives a global network of gene exchange connecting the human microbiome},  journal = {Nature}  }" data-bib-key="Smillie_2011" contenteditable="false">Smillie 2011), which reported an specificity of ARGs with analyzed environments, with a high degree of intra-habitat mobility of ARGs and few probabilities of genetic ARG transfer between different environments. 

In environments. 

In  this context, different factors such as MGEs (specially in flanking regions), founder effect, ecological connectivity between donor and acceptor, fitness cost or second-order selection may limit the gene transfer of ARGs between environments ( ...
Carrying the {blaDHA}-1 Gene and Its Regulator Gene {ampR},  Originated from Morganella morganii},  journal = {Antimicrobial Agents and Chemotherapy}  }" data-bib-key="Verdet_2000" contenteditable="false">Verdet 2000).

In 2000).

In  the same way that in our study, Fondi et al (2016) found that geography does not influence the microbial gene pool distributions, indicating that the dispersal potential of microorganisms is affected by environmental factors more than by geographical distances.

In 

In  order to analyze the grade of similarity between the b-lactamases detected in this study and the b-lactamases of clinical origin, present in the EX-B database, we choose four clinical important b-lactamases such blaTEM, blaCTX-M, blaGES (class A b-lactamases) and blaOXA (class D). Our results show that the distributions of those genes is different in the analyzed environments; thus, blaOXA was the most abundant gene, with a thousand of hits in soils, glaciers, fresh water, wastewater and human gut metagenomes. In the whole, blaOXA similarity was low, with 43.7 to 69.9% of hits showing a similarity between 50 to 59% to blaOXA sequences harbored in EX-B database; however, particularly interesting is the the human gut metagenome show a 24.3% of hits with high similarty (90 to 100% of similarity) to the blaOXA genes of the EX-B database. In addition; some differences were observed between the abundance of blaOXA gene in soil metagenomes; thus, non-agricultural metagenomes show a higher abundance (6480 hits) than the abundance observed in agricultural soils (3147 hits). It is clear that anthropogenic forces impact in different ways the b-lactamase content across the analyzed environments, since the abundance can be high in very impacted environments such as wastewater, but also can be high in less impacted environments such glaciers.

Similarly glaciers.

Similarly  to blaOXA, the other selected genes show the same trend, a very low percent of similarity to sequences in the EX-B database. This is particularly right in the case of soils, glaciers, fresh water and wastewater environments, but some differences can be observed in the distribution of the those genes across the different environments. for example a higher similarity to sequences in the EX-B database is observed in the case of blaCTX-M gene, with many hits classified in the range of 60 to 79 % of similarity, and more hits classified with 90 to 100% of similarity in the case of blaTEM (glaciers, fresh water and wastewater environmnets). 

In some cases few hits were obtained (less than 10) and the results need to be consider in accordance; thus one hit of blaCTX-M was obtained in among the rumen cow metagenomes, with a similarity classified in the range on 70 to 79%. In the opposite side, blaTEM exhibit 1335 hits among the human gut metagenomes; however, 1196 of them were obtained from a specific metagenome (human gut 8) and 113 of them show a 100% of similarity to blaTEM variants in the EX-B database. Certainly the two described examples are outliers, but in the case of the human gut metagenome, probably we are facing a sick person case, although more information about this metagenome is required to validate this hypothesis.  Low similarities between enviromental ARG and antibiotic resistance databases have been previously reported. Thus, in a metagenomic study,