A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics

Abstract

Pharmaceutical products contaminated with Burkholderia cepacia complex (BCC) strains constitute a serious health issue for susceptible individuals. New detection methods to distinguish DNA from viable cells are required to ensure pharmaceutical product quality and safety. In this study, we have assessed a droplet digital PCR (ddPCR) with a variant propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in autoclaved nuclease-free water after 365 days, in 0.001% chlorhexidine gluconate (CHX), and in 0.005% benzalkonium chloride (BZK) solutions after 184 days. Using 10 μM PMAxx and 5 min light exposure, a proportion of dead BCC was quantified by ddPCR. The detection limit of culture-based method was 104 CFU/mL, equivalent to 9.7 pg/μL for B. cenocepacia J2315, while that of ddPCR was 9.7 fg/μL. The true positive rate from nuclease-free water and CHX using PMAxx-ddPCR assay was 60.0% and 38.3%, respectively, compared to 85.0% and 74.6% without PMAxx (p < 0.05), respectively. However, in BZK-treated cells, no difference in the detection rate was observed between the ddPCR assay on samples treated with PMAxx (67.1%) and without PMAxx (63.3%). This study shows that the PMAxx-ddPCR assay provides a better tool for selective detection of live BCC cells in non-sterile pharmaceutical products.

Pilot clinical trial: propidium monoazide PCR quantifies reduction of the viable bacterial load after antiseptic preparation of canine oral mucosa

“…To further enhance sensitivity, the use of other molecular techniques combined with PMA may be considered for low-bacterial load environments. Gaoh et al (2022) found that PMA combined with droplet digital PCR was more sensitive than culture in detecting a viable pathogen in pharmaceutical products. 14 Droplet digital PCR is a newer molecular technique that allows for the detection of sequencespecific PCR products in potentially tens of thousands of micro-reactions.…”

“…Gaoh et al (2022) found that PMA combined with droplet digital PCR was more sensitive than culture in detecting a viable pathogen in pharmaceutical products. 14 Droplet digital PCR is a newer molecular technique that allows for the detection of sequencespecific PCR products in potentially tens of thousands of micro-reactions. This method offers several advantages including increased sensitivity and specificity even in low-bacterial load environments, as well as absolute quantification.…”

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 ].…”

Rapid Monitoring of Viable Genetically Modified Escherichia coli Using a Cell-Direct Quantitative PCR Method Combined with Propidium Monoazide Treatment

“…Such occurrences can pose a significant hazard. Conventional polymerase chain reaction (PCR), nested PCR, real-time quantitative PCR (qPCR), droplet digital PCR (ddPCR), and next-generation sequencing have been widely used to detect and quantify GMMs [ 1 , 4 , 5 , 6 , 7 , 8 , 9 ]. The success of these methods depends primarily on the isolation of DNA.…”