Cargando…

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological impo...

Descripción completa

Detalles Bibliográficos
Autores principales: Maritz, Julia M., Rogers, Krysta H., Rock, Tara M., Liu, Nicole, Joseph, Susan, Land, Kirkwood M., Carlton, Jane M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653731/
https://www.ncbi.nlm.nih.gov/pubmed/28540488
http://dx.doi.org/10.1007/s00248-017-0996-9
_version_ 1783273263828303872
author Maritz, Julia M.
Rogers, Krysta H.
Rock, Tara M.
Liu, Nicole
Joseph, Susan
Land, Kirkwood M.
Carlton, Jane M.
author_facet Maritz, Julia M.
Rogers, Krysta H.
Rock, Tara M.
Liu, Nicole
Joseph, Susan
Land, Kirkwood M.
Carlton, Jane M.
author_sort Maritz, Julia M.
collection PubMed
description Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00248-017-0996-9) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5653731
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher Springer US
record_format MEDLINE/PubMed
spelling pubmed-56537312017-11-01 An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads Maritz, Julia M. Rogers, Krysta H. Rock, Tara M. Liu, Nicole Joseph, Susan Land, Kirkwood M. Carlton, Jane M. Microb Ecol Methods Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00248-017-0996-9) contains supplementary material, which is available to authorized users. Springer US 2017-05-24 2017 /pmc/articles/PMC5653731/ /pubmed/28540488 http://dx.doi.org/10.1007/s00248-017-0996-9 Text en © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Methods
Maritz, Julia M.
Rogers, Krysta H.
Rock, Tara M.
Liu, Nicole
Joseph, Susan
Land, Kirkwood M.
Carlton, Jane M.
An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads
title An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads
title_full An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads
title_fullStr An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads
title_full_unstemmed An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads
title_short An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads
title_sort 18s rrna workflow for characterizing protists in sewage, with a focus on zoonotic trichomonads
topic Methods
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653731/
https://www.ncbi.nlm.nih.gov/pubmed/28540488
http://dx.doi.org/10.1007/s00248-017-0996-9
work_keys_str_mv AT maritzjuliam an18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT rogerskrystah an18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT rocktaram an18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT liunicole an18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT josephsusan an18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT landkirkwoodm an18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT carltonjanem an18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT maritzjuliam 18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT rogerskrystah 18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT rocktaram 18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT liunicole 18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT josephsusan 18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT landkirkwoodm 18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads
AT carltonjanem 18srrnaworkflowforcharacterizingprotistsinsewagewithafocusonzoonotictrichomonads