Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices
Autor(a) principal: | |
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Data de Publicação: | 2022 |
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/10451/53862 |
Resumo: | Mycobacterium bovis causes tuberculosis (TB) at the human-wildlife-livestock interface. Environmental persistence of M. bovis excreted by infected hosts may cause indirect transmission to other animals. However, methodological constrains hamper assessment of M. bovis viability and molecular signature in environmental matrices. In this work, an innovative, modular, and highly efficient single-cell workflow combining flow cytometry (FLOW), fluorescence in situ hybridization (FISH), and fluorescence-activated cell sorting (FACS) was developed, allowing detection, quantification, and sorting of viable and dormant M. bovis cells from environmental matrices. Validation with spiked water and sediments showed high efficiency (90%) of cell recovery, with high linearity between expected and observed results, both in cell viability evaluation (r2 =0.93) and FISH-labelled M. bovis cells quantification (r2 ≥0.96). The limit of detection was established at 105 cells/g of soil in the cell viability step and 102 cells/g of soil in the taxonomical labelling stage. Moreover, FACS efficiency attained noteworthy recovery yield (50%) and purity (60% viable cells; 70% taxonomically labelled M. bovis). This new methodology represents a huge step for M. bovis assessment outside the mammal host, offering the rapid quantification of M. bovis cell load and cell viability, including viable but non-culturable cells, and further downstream cell analyses after FACS. Subsequent environmental data integration with the clinical component will expand knowledge on transmission routes, promising new paths in TB research and an intervention tool to mitigate the underlying biohazard. |
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Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matricesMycobacterium bovis causes tuberculosis (TB) at the human-wildlife-livestock interface. Environmental persistence of M. bovis excreted by infected hosts may cause indirect transmission to other animals. However, methodological constrains hamper assessment of M. bovis viability and molecular signature in environmental matrices. In this work, an innovative, modular, and highly efficient single-cell workflow combining flow cytometry (FLOW), fluorescence in situ hybridization (FISH), and fluorescence-activated cell sorting (FACS) was developed, allowing detection, quantification, and sorting of viable and dormant M. bovis cells from environmental matrices. Validation with spiked water and sediments showed high efficiency (90%) of cell recovery, with high linearity between expected and observed results, both in cell viability evaluation (r2 =0.93) and FISH-labelled M. bovis cells quantification (r2 ≥0.96). The limit of detection was established at 105 cells/g of soil in the cell viability step and 102 cells/g of soil in the taxonomical labelling stage. Moreover, FACS efficiency attained noteworthy recovery yield (50%) and purity (60% viable cells; 70% taxonomically labelled M. bovis). This new methodology represents a huge step for M. bovis assessment outside the mammal host, offering the rapid quantification of M. bovis cell load and cell viability, including viable but non-culturable cells, and further downstream cell analyses after FACS. Subsequent environmental data integration with the clinical component will expand knowledge on transmission routes, promising new paths in TB research and an intervention tool to mitigate the underlying biohazard.ElsevierRepositório da Universidade de LisboaPereira, André C.Tenreiro, AnaTenreiro, RogérioCunha, Mónica V.2022-062024-06-01T00:00:00Z2022-06-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10451/53862eng10.1016/j.jhazmat.2022.128687info:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-08T17:00:05Zoai:repositorio.ul.pt:10451/53862Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T22:04:50.494129Repositório 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 |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices |
title |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices |
spellingShingle |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices Pereira, André C. |
title_short |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices |
title_full |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices |
title_fullStr |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices |
title_full_unstemmed |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices |
title_sort |
Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices |
author |
Pereira, André C. |
author_facet |
Pereira, André C. Tenreiro, Ana Tenreiro, Rogério Cunha, Mónica V. |
author_role |
author |
author2 |
Tenreiro, Ana Tenreiro, Rogério Cunha, Mónica V. |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
Repositório da Universidade de Lisboa |
dc.contributor.author.fl_str_mv |
Pereira, André C. Tenreiro, Ana Tenreiro, Rogério Cunha, Mónica V. |
description |
Mycobacterium bovis causes tuberculosis (TB) at the human-wildlife-livestock interface. Environmental persistence of M. bovis excreted by infected hosts may cause indirect transmission to other animals. However, methodological constrains hamper assessment of M. bovis viability and molecular signature in environmental matrices. In this work, an innovative, modular, and highly efficient single-cell workflow combining flow cytometry (FLOW), fluorescence in situ hybridization (FISH), and fluorescence-activated cell sorting (FACS) was developed, allowing detection, quantification, and sorting of viable and dormant M. bovis cells from environmental matrices. Validation with spiked water and sediments showed high efficiency (90%) of cell recovery, with high linearity between expected and observed results, both in cell viability evaluation (r2 =0.93) and FISH-labelled M. bovis cells quantification (r2 ≥0.96). The limit of detection was established at 105 cells/g of soil in the cell viability step and 102 cells/g of soil in the taxonomical labelling stage. Moreover, FACS efficiency attained noteworthy recovery yield (50%) and purity (60% viable cells; 70% taxonomically labelled M. bovis). This new methodology represents a huge step for M. bovis assessment outside the mammal host, offering the rapid quantification of M. bovis cell load and cell viability, including viable but non-culturable cells, and further downstream cell analyses after FACS. Subsequent environmental data integration with the clinical component will expand knowledge on transmission routes, promising new paths in TB research and an intervention tool to mitigate the underlying biohazard. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-06 2022-06-01T00:00:00Z 2024-06-01T00:00:00Z |
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/10451/53862 |
url |
http://hdl.handle.net/10451/53862 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1016/j.jhazmat.2022.128687 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/embargoedAccess |
eu_rights_str_mv |
embargoedAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Elsevier |
publisher.none.fl_str_mv |
Elsevier |
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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 |
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1799134599591231488 |