Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC)...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1094877/full |
| _version_ | 1811175497795633152 |
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| author | Muhammad Atif Nisar Kirstin E. Ross Melissa H. Brown Richard Bentham Giles Best Giles Best Harriet Whiley |
| author_facet | Muhammad Atif Nisar Kirstin E. Ross Melissa H. Brown Richard Bentham Giles Best Giles Best Harriet Whiley |
| author_sort | Muhammad Atif Nisar |
| collection | DOAJ |
| description | Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires’ disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a “viability based flow cytometry-cell sorting and qPCR” (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires’ disease. |
| first_indexed | 2024-04-10T19:36:56Z |
| format | Article |
| id | doaj.art-bd679ca062774bbab63b7016da62e642 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-10T19:36:56Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-bd679ca062774bbab63b7016da62e6422023-01-30T04:51:24ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-01-011410.3389/fmicb.2023.10948771094877Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCRMuhammad Atif Nisar0Kirstin E. Ross1Melissa H. Brown2Richard Bentham3Giles Best4Giles Best5Harriet Whiley6College of Science and Engineering, Flinders University, Bedford Park, SA, AustraliaCollege of Science and Engineering, Flinders University, Bedford Park, SA, AustraliaCollege of Science and Engineering, Flinders University, Bedford Park, SA, AustraliaCollege of Science and Engineering, Flinders University, Bedford Park, SA, AustraliaCollege of Medicine and Public Health, Flinders University, Bedford Park, SA, AustraliaFlow Cytometry Facility, Flinders University, Bedford Park, SA, AustraliaCollege of Science and Engineering, Flinders University, Bedford Park, SA, AustraliaLegionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires’ disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a “viability based flow cytometry-cell sorting and qPCR” (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires’ disease.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1094877/fullLegionnaires’ diseasepremise plumbingengineered water systempotable waterISO11731:2017-05ISO/TS12869:2019 |
| spellingShingle | Muhammad Atif Nisar Kirstin E. Ross Melissa H. Brown Richard Bentham Giles Best Giles Best Harriet Whiley Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR Frontiers in Microbiology Legionnaires’ disease premise plumbing engineered water system potable water ISO11731:2017-05 ISO/TS12869:2019 |
| title | Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR |
| title_full | Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR |
| title_fullStr | Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR |
| title_full_unstemmed | Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR |
| title_short | Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR |
| title_sort | detection and quantification of viable but non culturable legionella pneumophila from water samples using flow cytometry cell sorting and quantitative pcr |
| topic | Legionnaires’ disease premise plumbing engineered water system potable water ISO11731:2017-05 ISO/TS12869:2019 |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1094877/full |
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