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Abstract
Importance
Introduction
Historically, b-lactam antibiotics are the most widely used antibiotics in clinical settings
( around the world ( class="ltx_cite" data-bib-text="@article{Garau_2005,
doi = {10.1128/aac.49.7.2778-2784.2005},
url = {http://dx.doi.org/10.1128/aac.49.7.2778-2784.2005},
year = 2005,
...
author = {G. Garau and A. M. Di Guilmi and B. G. Hall},
title = {Structure-Based Phylogeny of the Metallo-~-Lactamases},
journal = {Antimicrobial Agents and Chemotherapy}
}" data-bib-key="Garau_2005" contenteditable="false">
Garau 2005,
class="ltx_cite" data-bib-text="@article{Van_Boeckel_2014,
doi = {10.1016/s1473-3099(14)70780-7},
url = {http://dx.doi.org/10.1016/s1473-3099(14)70780-7},
year = 2014,
...
author = {Thomas P Van Boeckel and Sumanth Gandra and Ashvin Ashok and Quentin Caudron and Bryan T Grenfell and Simon A Levin and Ramanan Laxminarayan},
title = {Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data},
journal = {The Lancet Infectious Diseases}
}" data-bib-key="Van_Boeckel_2014" contenteditable="false">
Boeckel 2014 ESAC 2009, ECDC 2012). The major cause of resistance to b-lactams antibiotics are bacterial enzymes with the capacity to hydrolyze the molecular structure of the b-lactam
antibiotic. antibiotic (Davies 1994). Many studies assessed the presence and diversity of b-lactamases in clinical settings
( ( class="ltx_cite" data-bib-text="@article{Paterson_2005,
doi = {10.1128/cmr.18.4.657-686.2005},
url = {http://dx.doi.org/10.1128/cmr.18.4.657-686.2005},
year = 2005,
...
author = {D. L. Paterson and R. A. Bonomo},
title = {Extended-Spectrum ~-Lactamases: a Clinical Update},
journal = {Clinical Microbiology Reviews}
}" data-bib-key="Paterson_2005" contenteditable="false">
Paterson 2005,
class="ltx_cite" data-bib-text="@article{Rice_2001,
doi = {10.1378/chest.119.2_suppl.391s},
url = {http://dx.doi.org/10.1378/chest.119.2_suppl.391s},
year = 2001,
...
author = {Louis Rice},
title = {Evolution and Clinical Importance of Extended-Spectrum $\upbeta$-Lactamases},
journal = {Chest}
}" data-bib-key="Rice_2001" contenteditable="false">
Rice 2001,
class="ltx_cite" data-bib-text="@article{Ramphal_2006,
doi = {10.1086/500663},
url = {http://dx.doi.org/10.1086/500663},
year = 2006,
...
author = {R. Ramphal and P. G. A. $\greater$},
title = {Extended-Spectrum ~-Lactamases and Clinical Outcomes: Current Data},
journal = {Clinical Infectious Diseases}
}" data-bib-key="Ramphal_2006" contenteditable="false">
Ramphal 2006,
class="ltx_cite" data-bib-text="@article{Shahid_2014,
doi = {10.12816/0008130},
url = {http://dx.doi.org/10.12816/0008130},
year = 2014,
...
author = {Mohammad Shahid},
title = {Prevalence of {CTX} M Extended-Spectrum Beta-Lactamases in Clinical Gram-Negative Bacteria},
journal = {Bahrain Medical Bulletin}
}" data-bib-key="Shahid_2014" contenteditable="false">
Shahid 2014,
class="ltx_cite" data-bib-text="@article{Sullivan_2015,
doi = {10.4172/2161-0703.1000203},
url = {http://dx.doi.org/10.4172/2161-0703.1000203},
year = 2015,
...
author = {Rebecca Sullivan and David Schaus},
title = {Extended Spectrum Beta- Lactamases: A Minireview of Clinical Relevant Groups},
journal = {Journal of Medical Microbiology {\&} Diagnosis}
}" data-bib-key="Sullivan_2015" contenteditable="false">
Sullivan 2015)
2015), but
only few no efforts have been put in assess
b-lactamases in non-clinical settings.
Few studies that evaluated the AR phenomenon in
other non-clinical environments
like soil, fresh water, or other (Amos 2014, Su 2014, Ma 2014). In this context metagenomic studies performed in the last years, have the capacity to achieve wide analyses of ARGs in environmental samples ( based on functional metagenomics studies, PCR reactions, MIC tests ( class="ltx_cite" data-bib-text="@article{Donato_2010,
doi = {10.1128/aem.01763-09},
url = {http://dx.doi.org/10.1128/aem.01763-09},
year = 2010,
...
author = {J. J. Donato and L. A. Moe and B. J. Converse and K. D. Smart and F. C. Berklein and P. S. McManus and J. Handelsman},
title = {Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins},
journal = {Applied and Environmental Microbiology}
}" data-bib-key="Donato_2010" contenteditable="false">
Donato 2010,
Yang 2013,
2010, class="ltx_cite" data-bib-text="@article{Bhullar_2012,
doi = {10.1371/journal.pone.0034953},
url = {http://dx.doi.org/10.1371/journal.pone.0034953},
year = 2012,
...
editor = {Ramy K. Aziz},
title = {Antibiotic Resistance Is Prevalent in an Isolated Cave Microbiome},
journal = {{PLoS} {ONE}}
}" data-bib-key="Bhullar_2012" contenteditable="false">
Bhullar 2012,
2012, class="ltx_cite" data-bib-text="@article{Segawa_2012,
doi = {10.1111/1758-2229.12011},
url = {http://dx.doi.org/10.1111/1758-2229.12011},
year = 2012,
...
author = {Takahiro Segawa and Nozomu Takeuchi and Andres Rivera and Akinori Yamada and Yoshitaka Yoshimura and Gonzalo Barcaza and Kunio Shinbori and Hideaki Motoyama and Shiro Kohshima and Kazunari Ushida},
title = {Distribution of antibiotic resistance genes in glacier environments},
journal = {Environmental Microbiology Reports}
}" data-bib-key="Segawa_2012" contenteditable="false">
Segawa 2012,
2012, Amos 2014, class="ltx_cite" data-bib-text="@article{Forsberg_2014,
doi = {10.1038/nature13377},
url = {http://dx.doi.org/10.1038/nature13377},
year = 2014,
...
author = {Kevin J. Forsberg and Sanket Patel and Molly K. Gibson and Christian L. Lauber and Rob Knight and Noah Fierer and Gautam Dantas},
title = {Bacterial phylogeny structures soil resistomes across habitats},
journal = {Nature}
}" data-bib-key="Forsberg_2014" contenteditable="false">
Forsberg 2014,
Su 2014, Ma 2014) and in the last years, the use of metagenomic approaches (Yang 2013, class="ltx_cite" data-bib-text="@article{Li_2015,
doi = {10.1038/ismej.2015.59},
url = {http://dx.doi.org/10.1038/ismej.2015.59},
year = 2015,
...
author = {Bing Li and Ying Yang and Liping Ma and Feng Ju and Feng Guo and James M Tiedje and Tong Zhang},
title = {Metagenomic and network analysis reveal wide distribution and co-occurrence of environmental antibiotic resistance genes},
journal = {The {ISME} Journal}
}" data-bib-key="Li_2015" contenteditable="false">
Li 2015,
2015, class="ltx_cite" data-bib-text="@article{Fondi_2016,
doi = {10.1093/gbe/evw077},
url = {http://dx.doi.org/10.1093/gbe/evw077},
year = 2016,
...
author = {Marco Fondi and Antti Karkman and Manu V. Tamminen and Emanuele Bosi and Marko Virta and Renato Fani and Eric Alm and James O. McInerney},
title = {{\textquotedblleft}Every Gene Is Everywhere but the Environment Selects{\textquotedblright}: Global Geolocalization of Gene Sharing in Environmental Samples through Network Analysis},
journal = {Genome Biol Evol}
}" data-bib-key="Fondi_2016" contenteditable="false">
Fondi 2016); thus, presence and diversity
2016) appear such as an interesting new 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
could shed light about both 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, no studies focused on b-lactamases have been performed until now, avoiding the understanding of important process with clinical implications such as b-lactamase gene transfer between environments and theanthropogenic impact of anthropogenic forces on b-lactamase content and diversity in natural environments.
The 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 environmenets.
Results
Discussion
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
( ( class="ltx_cite" data-bib-text="@article{Aminov_2009,
doi = {10.1111/j.1462-2920.2009.01972.x},
url = {http://dx.doi.org/10.1111/j.1462-2920.2009.01972.x},
year = 2009,
...
author = {Rustam I. Aminov},
title = {The role of antibiotics and antibiotic resistance in nature},
journal = {Environmental Microbiology}
}" data-bib-key="Aminov_2009" contenteditable="false">
href="#Aminov_2009">Aminov href="https://www.authorea.com/users/70389/articles/118942/_show_article#Aminov_2009">Aminov 2009,
class="ltx_cite" data-bib-text="@article{Hall_2004,
doi = {10.1016/j.drup.2004.02.003},
url = {http://dx.doi.org/10.1016/j.drup.2004.02.003},
year = 2004,
...
title = {Evolution of the serine $\upbeta$-lactamases: past,
present and future},
journal = {Drug Resistance Updates}
}" data-bib-key="Hall_2004" contenteditable="false">
href="#Hall_2004">Hall href="https://www.authorea.com/users/70389/articles/118942/_show_article#Hall_2004">Hall 2004,
class="ltx_cite" data-bib-text="@article{Garau_2005,
doi = {10.1128/aac.49.7.2778-2784.2005},
url = {http://dx.doi.org/10.1128/aac.49.7.2778-2784.2005},
year = 2005,
...
author = {G. Garau and A. M. Di Guilmi and B. G. Hall},
title = {Structure-Based Phylogeny of the Metallo-~-Lactamases},
journal = {Antimicrobial Agents and Chemotherapy}
}" data-bib-key="Garau_2005" contenteditable="false">
href="#Garau_2005">Garau href="https://www.authorea.com/users/70389/articles/118942/_show_article#Garau_2005">Garau 2005). Thus, natural environments appear such as a reservoir of potential ARGs to pathogenic bacteria
( ( class="ltx_cite" data-bib-text="@article{Berglund_2015,
doi = {10.3402/iee.v5.28564},
url = {http://dx.doi.org/10.3402/iee.v5.28564},
year = 2015,
...
author = {Björn Berglund},
title = {Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with antibiotics},
journal = {Infection Ecology {\&} Epidemiology}
}" data-bib-key="Berglund_2015" contenteditable="false">
href="#Berglund_2015">Berglund href="https://www.authorea.com/users/70389/articles/118942/_show_article#Berglund_2015">Berglund 2015,
class="ltx_cite" data-bib-text="@article{Versluis_2015,
doi = {10.1038/srep11981},
url = {http://dx.doi.org/10.1038/srep11981},
year = 2015,
...
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">
href="#Versluis_2015">Versluis href="https://www.authorea.com/users/70389/articles/118942/_show_article#Versluis_2015">Versluis 2015) that cannot be
ignored.
Results
Discussion
Materials ignored.
Materials and methods
Data set
Shotgun metagenomic sequences obtained by Illumina sequencing process were used in this study and downloaded from two repositories, MG-RAST (http://metagenomics.anl.gov/) and EBI METAGENOMICS (https://www.ebi.ac.uk/metagenomics/;jsessionid=5B7CC5F7C4C8CD7C9FB5F7E8A4D1C621). A total of 232 metagenomes related to X different projects (Table S1) embedding 4.7 billion sequences, were retrieved after quality control steps performed by each repositorie. Each metagenome was associated with different sampling habitats including soil (undisturbed and agricultural soils), fresh water, ocean, glaciers, human gut, animal gut (rumen and feces), and effluents from wastewater treatment plants.
Construction of a comprehensive β-lactamases
database
Characterization of metagenome sequences associated to β-lactamase genes was preceded by construction of an
extensive β-lactamase database (EX-B) that integrated four clinically-important
publically-available databases: The Lahey β-lactamase database,
...
OXY, OKP and LEN protein variation databases (Gatica et al., 2016). Sequences deposited into EX-B were compared using
BioEdit 7.2.5 software and checked for non-redundant sequences; producing a
database containing 1566 non-redundant β-lactamase sequences. The EX-B
database is available for download at (http://app.agri.gov.il/eddie/tools.html).
Blast searches
Homology searches among metagenomic and b-lactamase sequences were performed using blastx from the BLAST suite ( ( class="ltx_cite" data-bib-text="@article{Gish_1993,
doi = {10.1038/ng0393-266},
url = {http://dx.doi.org/10.1038/ng0393-266},
year = 1993,
...
author = {Warren Gish and David J. States},
title = {Identification of protein coding regions by database similarity search},
journal = {Nature Genetics}
}" data-bib-key="Gish_1993" contenteditable="false">Gish 1993). Only hits with a percent of identity higher than 50%, bit score higher than 30 and a e-value lower than 1e-4 were considered real hits. The 50% of identity was used under the consideration that the databases used to construct the EX-B database are highly based in clinical data and generally it not consider environmental data.
Hits analysis
Hits obtained by BLAST were analyzed in PC-ORD 5.0 (McCune and Mefford, 2011).The hits were used to construct a matrix of present hits, according b-lactamase gene type, in each metagenome. The data was relativizated by weighting by ubiquity and transformed by square root. Outliers were identified and removed of analysis and a distance matrix based on Bray-Curtis distance was constructed to downstream analysis. In addition, the distance matrix constructed was used to obtain diversity indexes, multi response permutation process (MRPP), indicator species analysis and non metric multidimensional scaling.
The Distance matrix from the previous step was used to construct a b-lactamase gene network using EDENetwork 2.18 ( ( class="ltx_cite" data-bib-text="@article{Kivel__2014,
doi = {10.1111/1755-0998.12290},
url = {http://dx.doi.org/10.1111/1755-0998.12290},
year = 2014,
...
title = {{EDENetworks}: A user-friendly software to build and analyse networks in biogeography,
ecology and population genetics},
journal = {Molecular Ecology Resources}
}" data-bib-key="Kivel__2014" contenteditable="false">Kivelä 2014). Graphical visualization and statistical test were performed using Cytoscape 3.4.0 ( ( class="ltx_cite" data-bib-text="@article{Shannon_2003,
doi = {10.1101/gr.1239303},
url = {http://dx.doi.org/10.1101/gr.1239303},
year = 2003,
