Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli.
Autor(a) principal: | |
---|---|
Data de Publicação: | 2009 |
Outros Autores: | , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/117176 |
Resumo: | BACKGROUND Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems. RESULTS EtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 +/- 0.0057 min-1; = 0.0106 +/- 0.0033 min-1; and = 0.0230 +/- 0.0075 min-1. CONCLUSION The combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall. |
id |
RCAP_3f9d2591b11d76013799100db0ff3423 |
---|---|
oai_identifier_str |
oai:run.unl.pt:10362/117176 |
network_acronym_str |
RCAP |
network_name_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository_id_str |
7160 |
spelling |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli.Biochemistry, Genetics and Molecular Biology (miscellaneous)MicrobiologyInfectious DiseasesSDG 3 - Good Health and Well-beingBACKGROUND Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems. RESULTS EtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 +/- 0.0057 min-1; = 0.0106 +/- 0.0033 min-1; and = 0.0230 +/- 0.0075 min-1. CONCLUSION The combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall.Instituto de Higiene e Medicina Tropical (IHMT)Unidade de Parasitologia e Microbiologia Médicas (UPMM)CREM - Centro de Recursos MicrobiológicosRUNPaixão, LauraRodrigues, LCouto, Isabel Maria dos Santos LeitãoMartins, MartaFernandes, Pedrode Carvalho, Carla CCRMonteiro, Gabriel ASansonetty, FilipeAmaral, LeonardViveiros, M2021-05-06T22:35:48Z2009-01-012009-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10362/117176eng1754-1611PURE: 294204https://doi.org/10.1186/1754-1611-3-18info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:00:16Zoai:run.unl.pt:10362/117176Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:43:31.198053Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. |
title |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. |
spellingShingle |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. Paixão, Laura Biochemistry, Genetics and Molecular Biology (miscellaneous) Microbiology Infectious Diseases SDG 3 - Good Health and Well-being |
title_short |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. |
title_full |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. |
title_fullStr |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. |
title_full_unstemmed |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. |
title_sort |
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. |
author |
Paixão, Laura |
author_facet |
Paixão, Laura Rodrigues, L Couto, Isabel Maria dos Santos Leitão Martins, Marta Fernandes, Pedro de Carvalho, Carla CCR Monteiro, Gabriel A Sansonetty, Filipe Amaral, Leonard Viveiros, M |
author_role |
author |
author2 |
Rodrigues, L Couto, Isabel Maria dos Santos Leitão Martins, Marta Fernandes, Pedro de Carvalho, Carla CCR Monteiro, Gabriel A Sansonetty, Filipe Amaral, Leonard Viveiros, M |
author2_role |
author author author author author author author author author |
dc.contributor.none.fl_str_mv |
Instituto de Higiene e Medicina Tropical (IHMT) Unidade de Parasitologia e Microbiologia Médicas (UPMM) CREM - Centro de Recursos Microbiológicos RUN |
dc.contributor.author.fl_str_mv |
Paixão, Laura Rodrigues, L Couto, Isabel Maria dos Santos Leitão Martins, Marta Fernandes, Pedro de Carvalho, Carla CCR Monteiro, Gabriel A Sansonetty, Filipe Amaral, Leonard Viveiros, M |
dc.subject.por.fl_str_mv |
Biochemistry, Genetics and Molecular Biology (miscellaneous) Microbiology Infectious Diseases SDG 3 - Good Health and Well-being |
topic |
Biochemistry, Genetics and Molecular Biology (miscellaneous) Microbiology Infectious Diseases SDG 3 - Good Health and Well-being |
description |
BACKGROUND Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems. RESULTS EtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 +/- 0.0057 min-1; = 0.0106 +/- 0.0033 min-1; and = 0.0230 +/- 0.0075 min-1. CONCLUSION The combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall. |
publishDate |
2009 |
dc.date.none.fl_str_mv |
2009-01-01 2009-01-01T00:00:00Z 2021-05-06T22:35:48Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/117176 |
url |
http://hdl.handle.net/10362/117176 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1754-1611 PURE: 294204 https://doi.org/10.1186/1754-1611-3-18 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
_version_ |
1799138044621619200 |