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

Abstract

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.

Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions

“…In previous studies, we identified 206 BCC-specific gene clusters which are only present in BCC genomes from a total of 174,715 clusters (i.e., orthologous gene groups with ≥75% sequence identity) of 266 complete genomes (i.e., 82 of BCC and 184 of non-BCC, respectively) of the genus Burkholderia [ 12 , 20 , 21 ]. In this study, among the 206 BCC-specific clusters, we chose a gene cluster (No.…”

Detection, Genophenotypic Characterization, and Antimicrobial Resistance of Microbial Contaminants

“…Understanding the risk factors associated with different microorganisms has led to improvements in controlling microbial contamination [ 1 , 2 ]. This Special Issue was established to gather and share high-quality scientific articles on microbial contamination and contains six original research articles covering a range of diverse topics related to microbial contamination [ 3 , 4 , 5 , 6 , 7 , 8 ]. As revealed from the retrospective analysis of the articles, microbial contamination challenges are often focused around three key issues—the detection, genophenotypic characterization, and antimicrobial resistance (AMR) of microbial contaminants, whereby all of which are interconnected in a pleiotropic and epistatic manner.…”

“…Detecting microbial contaminants in samples with low biomass and complex matrices is a challenging task that requires rapid, sensitive, and accurate detection methods for effective safety interventions. In this Special Issue, several articles explore ways to overcome these challenges and improve microbial detection outcomes [ 3 , 4 , 5 , 6 , 8 ].…”

“…The Burkholderia cepacia complex (BCC) is a group of closely related pathogenic Burkholderia species that can contaminate non-sterilized pharmaceutical materials and long-term water-based products, posing a potential risk to public health [ 5 , 6 ]. In their articles, Daddy Gaoh et al have presented two new molecular-based methods for the selective detection of live BCC in different materials: a flow-cytometry-based detection method using a fluorescently labeled oligonucleotide Kef probe and a droplet digital PCR (ddPCR) with a variant of propidium monoazide (PMAxx) [ 5 , 6 ]. The study noted that designing BCC-specific primers or probes is challenging due to the close genomic and phenomic relatedness of BCC with other non-BCC strains.…”

“…The study noted that designing BCC-specific primers or probes is challenging due to the close genomic and phenomic relatedness of BCC with other non-BCC strains. The challenge of designing BCC-specific primers or probes was overcome by adopting a pan-genome-based bioinformatics pipeline to analyze complete Burkholderia genomes and identify BCC-specific gene clusters that are only present in BCC genomes [ 5 , 6 ]. Additionally, the research group combined PMAxx with ddPCR to specifically detect viable BCC cells, addressing a main disadvantage of molecular-based methods that can lead to false positive results [ 6 ].…”