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Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe

Burkholderia cepacia complex (BCC) contamination has resulted in recalls of non-sterile pharmaceutical products. The fast, sensitive, and specific detection of BCC is critical for ensuring the quality and safety of pharmaceutical products. In this study, a rapid flow cytometry-based detection method...

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Autores principales: Daddy Gaoh, Soumana, Williams, Anna, Le, David, Kweon, Ohgew, Alusta, Pierre, Buzatu, Dan A., Ahn, Youngbeom
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9227203/
https://www.ncbi.nlm.nih.gov/pubmed/35744688
http://dx.doi.org/10.3390/microorganisms10061170
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author Daddy Gaoh, Soumana
Williams, Anna
Le, David
Kweon, Ohgew
Alusta, Pierre
Buzatu, Dan A.
Ahn, Youngbeom
author_facet Daddy Gaoh, Soumana
Williams, Anna
Le, David
Kweon, Ohgew
Alusta, Pierre
Buzatu, Dan A.
Ahn, Youngbeom
author_sort Daddy Gaoh, Soumana
collection PubMed
description Burkholderia cepacia complex (BCC) contamination has resulted in recalls of non-sterile pharmaceutical products. The fast, sensitive, and specific detection of BCC is critical for ensuring the quality and safety of pharmaceutical products. In this study, a rapid flow cytometry-based detection method was developed using a fluorescence-labeled oligonucleotide Kef probe that specifically binds a KefB/KefC membrane protein sequence within BCC. Optimal conditions of a 1 nM Kef probe concentration at a 60 °C hybridization temperature for 30 min were determined and applied for the flow cytometry assay. The true-positive rate (sensitivity) and true-negative rate (specificity) of the Kef probe assay were 90% (18 positive out of 20 BCC species) and 88.9% (16 negative out of 18 non-BCC), respectively. The detection limit for B. cenocepacia AU1054 with the Kef probe flow cytometry assay in nuclease-free water was 1 CFU/mL. The average cell counts using the Kef probe assay from a concentration of 10 μg/mL chlorhexidine gluconate and 50 μg/mL benzalkonium chloride were similar to those of the RAPID-B total plate count (TPC). We demonstrate the potential of Kef probe flow cytometry as a more sensitive alternative to culture-based methods for detecting BCC in non-sterilized pharmaceutical raw materials and products with regards to water-based environments.
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spelling pubmed-92272032022-06-25 Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe Daddy Gaoh, Soumana Williams, Anna Le, David Kweon, Ohgew Alusta, Pierre Buzatu, Dan A. Ahn, Youngbeom Microorganisms Article Burkholderia cepacia complex (BCC) contamination has resulted in recalls of non-sterile pharmaceutical products. The fast, sensitive, and specific detection of BCC is critical for ensuring the quality and safety of pharmaceutical products. In this study, a rapid flow cytometry-based detection method was developed using a fluorescence-labeled oligonucleotide Kef probe that specifically binds a KefB/KefC membrane protein sequence within BCC. Optimal conditions of a 1 nM Kef probe concentration at a 60 °C hybridization temperature for 30 min were determined and applied for the flow cytometry assay. The true-positive rate (sensitivity) and true-negative rate (specificity) of the Kef probe assay were 90% (18 positive out of 20 BCC species) and 88.9% (16 negative out of 18 non-BCC), respectively. The detection limit for B. cenocepacia AU1054 with the Kef probe flow cytometry assay in nuclease-free water was 1 CFU/mL. The average cell counts using the Kef probe assay from a concentration of 10 μg/mL chlorhexidine gluconate and 50 μg/mL benzalkonium chloride were similar to those of the RAPID-B total plate count (TPC). We demonstrate the potential of Kef probe flow cytometry as a more sensitive alternative to culture-based methods for detecting BCC in non-sterilized pharmaceutical raw materials and products with regards to water-based environments. MDPI 2022-06-07 /pmc/articles/PMC9227203/ /pubmed/35744688 http://dx.doi.org/10.3390/microorganisms10061170 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Daddy Gaoh, Soumana
Williams, Anna
Le, David
Kweon, Ohgew
Alusta, Pierre
Buzatu, Dan A.
Ahn, Youngbeom
Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe
title Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe
title_full Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe
title_fullStr Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe
title_full_unstemmed Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe
title_short Specific Detection and Enumeration of Burkholderia cepacia Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe
title_sort specific detection and enumeration of burkholderia cepacia complex by flow cytometry using a fluorescence-labeled oligonucleotide probe
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9227203/
https://www.ncbi.nlm.nih.gov/pubmed/35744688
http://dx.doi.org/10.3390/microorganisms10061170
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