A functional metagenomic analysis of tetracycline resistance in cheese bacteria

Metagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate...

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Autores: Flórez, Ana Belén, Vázquez, Lucía, Mayo Pérez, Baltasar
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2017
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/184931
Acceso en línea:http://hdl.handle.net/10261/184931
Access Level:acceso abierto
Palabra clave:Antibiotic resistance
Resistance genes
Tetracycline resistance
Cheeses
Traditional cheeses
Culture independent methods
id ES_8f26dda24caa2f00a538242e12b3548b
oai_identifier_str oai:digital.csic.es:10261/184931
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv A functional metagenomic analysis of tetracycline resistance in cheese bacteria
title A functional metagenomic analysis of tetracycline resistance in cheese bacteria
spellingShingle A functional metagenomic analysis of tetracycline resistance in cheese bacteria
Flórez, Ana Belén
Antibiotic resistance
Resistance genes
Tetracycline resistance
Cheeses
Traditional cheeses
Culture independent methods
title_short A functional metagenomic analysis of tetracycline resistance in cheese bacteria
title_full A functional metagenomic analysis of tetracycline resistance in cheese bacteria
title_fullStr A functional metagenomic analysis of tetracycline resistance in cheese bacteria
title_full_unstemmed A functional metagenomic analysis of tetracycline resistance in cheese bacteria
title_sort A functional metagenomic analysis of tetracycline resistance in cheese bacteria
dc.creator.none.fl_str_mv Flórez, Ana Belén
Vázquez, Lucía
Mayo Pérez, Baltasar
author Flórez, Ana Belén
author_facet Flórez, Ana Belén
Vázquez, Lucía
Mayo Pérez, Baltasar
author_role author
author2 Vázquez, Lucía
Mayo Pérez, Baltasar
author2_role author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
Principado de Asturias
Mayo Pérez, Baltasar [0000-0001-5634-6543]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Antibiotic resistance
Resistance genes
Tetracycline resistance
Cheeses
Traditional cheeses
Culture independent methods
topic Antibiotic resistance
Resistance genes
Tetracycline resistance
Cheeses
Traditional cheeses
Culture independent methods
description Metagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. The present work reports a functional metagenomic analysis of the prevalence and evolution of tetracycline resistance determinants in a raw-milk, blue-veined cheese during manufacturing and ripening. For this, the same cheese batch was sampled and analyzed on days 3 and 60 of manufacture. Samples were diluted and grown in the presence of tetracycline on plate count milk agar (PCMA) (non-selective) and de Man Rogosa and Sharpe (MRS) agar (selective for lactic acid bacteria, LAB). DNA from the cultured bacteria was then isolated and used to construct four fosmid libraries, named after the medium and sampling time: PCMA-3D, PCMA-60D, MRS-3D, and MRS-60D. Clones in the libraries were subjected to restriction enzyme analysis, PCR amplification, and sequencing. Among the 300 fosmid clones analyzed, 268 different EcoRI restriction profiles were encountered. Sequence homology of their extremes clustered the clones into 47 groups. Representative clones of all groups were then screened for the presence of tetracycline resistance genes by PCR, targeting well-recognized genes coding for ribosomal protection proteins and efflux pumps. A single tetracycline resistance gene was detected in each of the clones, with four such resistance genes identified in total: tet(A), tet(L), tet(M), and tet(S). tet(A) was the only gene identified in the PCMA-3D library, and tet(L) the only one identified in the PCMA-60D and MRS-60D libraries. tet(M) and tet(S) were both detected in the MRS-3D library and in similar numbers. Six representative clones of the libraries were sequenced and analyzed. Long segments of all clones but one showed extensive homology to plasmids from Gram-positive and Gram-negative bacteria. tet(A) was found within a sequence showing strong similarity to plasmids pMAK2 and pO26-Vir from Salmonella enterica and Escherichia coli, respectively. All other genes were embedded in, or near to, sequences homologous to those of LAB species. These findings strongly suggest an evolution of tetracycline resistance gene types during cheese ripening, which might reflect the succession of the microbial populations. The location of the tetracycline resistance genes in plasmids, surrounded or directly flanked by open reading frames encoding transposases, invertases or mobilization proteins, suggests they might have a strong capacity for transference. Raw-milk cheeses should therefore be considered reservoirs of tetracycline resistance genes that might be horizontally transferred.
publishDate 2017
dc.date.none.fl_str_mv 2017
2019
2019
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/184931
url http://hdl.handle.net/10261/184931
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/AGL-2014-57820-R
http://dx.doi.org/10.3389/fmicb.2017.00907
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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spelling A functional metagenomic analysis of tetracycline resistance in cheese bacteriaFlórez, Ana BelénVázquez, LucíaMayo Pérez, BaltasarAntibiotic resistanceResistance genesTetracycline resistanceCheesesTraditional cheesesCulture independent methodsMetagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. The present work reports a functional metagenomic analysis of the prevalence and evolution of tetracycline resistance determinants in a raw-milk, blue-veined cheese during manufacturing and ripening. For this, the same cheese batch was sampled and analyzed on days 3 and 60 of manufacture. Samples were diluted and grown in the presence of tetracycline on plate count milk agar (PCMA) (non-selective) and de Man Rogosa and Sharpe (MRS) agar (selective for lactic acid bacteria, LAB). DNA from the cultured bacteria was then isolated and used to construct four fosmid libraries, named after the medium and sampling time: PCMA-3D, PCMA-60D, MRS-3D, and MRS-60D. Clones in the libraries were subjected to restriction enzyme analysis, PCR amplification, and sequencing. Among the 300 fosmid clones analyzed, 268 different EcoRI restriction profiles were encountered. Sequence homology of their extremes clustered the clones into 47 groups. Representative clones of all groups were then screened for the presence of tetracycline resistance genes by PCR, targeting well-recognized genes coding for ribosomal protection proteins and efflux pumps. A single tetracycline resistance gene was detected in each of the clones, with four such resistance genes identified in total: tet(A), tet(L), tet(M), and tet(S). tet(A) was the only gene identified in the PCMA-3D library, and tet(L) the only one identified in the PCMA-60D and MRS-60D libraries. tet(M) and tet(S) were both detected in the MRS-3D library and in similar numbers. Six representative clones of the libraries were sequenced and analyzed. Long segments of all clones but one showed extensive homology to plasmids from Gram-positive and Gram-negative bacteria. tet(A) was found within a sequence showing strong similarity to plasmids pMAK2 and pO26-Vir from Salmonella enterica and Escherichia coli, respectively. All other genes were embedded in, or near to, sequences homologous to those of LAB species. These findings strongly suggest an evolution of tetracycline resistance gene types during cheese ripening, which might reflect the succession of the microbial populations. The location of the tetracycline resistance genes in plasmids, surrounded or directly flanked by open reading frames encoding transposases, invertases or mobilization proteins, suggests they might have a strong capacity for transference. Raw-milk cheeses should therefore be considered reservoirs of tetracycline resistance genes that might be horizontally transferred.The study was partially supported by projects from the Spanish Ministry of Economy and Competitiveness (AGL-2014-57820-R) and Asturias Principality (GRUPIN14-137). AF and LV were supported by research contracts of the JAE-Doc Program from CSIC and the FPI Program from MINECO (BES-2015-072285), respectivelyPeer reviewedFrontiers MediaMinisterio de Economía y Competitividad (España)Principado de AsturiasMayo Pérez, Baltasar [0000-0001-5634-6543]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201920192017info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/184931reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/AGL-2014-57820-Rhttp://dx.doi.org/10.3389/fmicb.2017.00907Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1849312026-05-22T06:33:51Z
score 15,811543

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