Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique
Very low cell count detection of Escherichia coli O157:H7 in foods is critical, since an infective dose for this pathogen may be only 10 cells, and fewer still for vulnerable populations. A flow cytometer is able to detect and count individual cells of a target bacterium, in this case E. coli O157:H...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2017-08-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/article/10.3389/fmicb.2017.01493/full |
| _version_ | 1818549133803257856 |
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| author | Anna J. Williams Willie M. Cooper Shawn Ramsaroop Pierre Alusta Dan A. Buzatu Jon G. Wilkes |
| author_facet | Anna J. Williams Willie M. Cooper Shawn Ramsaroop Pierre Alusta Dan A. Buzatu Jon G. Wilkes |
| author_sort | Anna J. Williams |
| collection | DOAJ |
| description | Very low cell count detection of Escherichia coli O157:H7 in foods is critical, since an infective dose for this pathogen may be only 10 cells, and fewer still for vulnerable populations. A flow cytometer is able to detect and count individual cells of a target bacterium, in this case E. coli O157:H7. The challenge is to find the single cell in a complex matrix like raw spinach. To find that cell requires growing it as quickly as possible to a number sufficiently in excess of matrix background that identification is certain. The experimental design for this work was that of a U.S. Food and Drug Administration (FDA) In-House Level 3 validation executed in the technology’s originating laboratory. Using non-selective enrichment broth, 6.5 h incubation at 42°C, centrifugation for target cell concentration, and a highly selective E. coli O157 fluorescent antibody tag, the cytometry method proved more sensitive than a reference regulatory method (p = 0.01) for detecting a single target cell, one E. coli O157:H7 cell, in 25 g of spinach. It counted that cell’s daughters with at least 38× signal-to-noise ratio, analyzing 25 samples in total-time-to-results of 9 h. |
| first_indexed | 2024-12-12T08:29:27Z |
| format | Article |
| id | doaj.art-565530b9f07f4604b06d08ddc972cb06 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-12T08:29:27Z |
| publishDate | 2017-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-565530b9f07f4604b06d08ddc972cb062022-12-22T00:31:09ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2017-08-01810.3389/fmicb.2017.01493278906Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation TechniqueAnna J. Williams0Willie M. Cooper1Shawn Ramsaroop2Pierre Alusta3Dan A. Buzatu4Jon G. Wilkes5Division of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, JeffersonAR, United StatesDivision of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, JeffersonAR, United StatesVivione Biosciences, LLC, Pine BluffAR, United StatesDivision of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, JeffersonAR, United StatesDivision of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, JeffersonAR, United StatesDivision of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, JeffersonAR, United StatesVery low cell count detection of Escherichia coli O157:H7 in foods is critical, since an infective dose for this pathogen may be only 10 cells, and fewer still for vulnerable populations. A flow cytometer is able to detect and count individual cells of a target bacterium, in this case E. coli O157:H7. The challenge is to find the single cell in a complex matrix like raw spinach. To find that cell requires growing it as quickly as possible to a number sufficiently in excess of matrix background that identification is certain. The experimental design for this work was that of a U.S. Food and Drug Administration (FDA) In-House Level 3 validation executed in the technology’s originating laboratory. Using non-selective enrichment broth, 6.5 h incubation at 42°C, centrifugation for target cell concentration, and a highly selective E. coli O157 fluorescent antibody tag, the cytometry method proved more sensitive than a reference regulatory method (p = 0.01) for detecting a single target cell, one E. coli O157:H7 cell, in 25 g of spinach. It counted that cell’s daughters with at least 38× signal-to-noise ratio, analyzing 25 samples in total-time-to-results of 9 h.http://journal.frontiersin.org/article/10.3389/fmicb.2017.01493/fullsample preparationfood pathogensbacterial detection in foodbacterial quantificationpublic health |
| spellingShingle | Anna J. Williams Willie M. Cooper Shawn Ramsaroop Pierre Alusta Dan A. Buzatu Jon G. Wilkes Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique Frontiers in Microbiology sample preparation food pathogens bacterial detection in food bacterial quantification public health |
| title | Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique |
| title_full | Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique |
| title_fullStr | Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique |
| title_full_unstemmed | Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique |
| title_short | Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique |
| title_sort | rapid flow cytometry detection of a single viable escherichia coli o157 h7 cell in raw spinach using a simplified sample preparation technique |
| topic | sample preparation food pathogens bacterial detection in food bacterial quantification public health |
| url | http://journal.frontiersin.org/article/10.3389/fmicb.2017.01493/full |
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