ABSTRACT
In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods. We assessed the groundwater microbiome's taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436-557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety.
Subject(s)
Bacteria , Groundwater , Metagenomics , Bacteria/genetics , Bacteria/classification , Bacteria/isolation & purification , Groundwater/microbiology , Microbiota/genetics , Pharmaceutical Preparations , Metagenome , Water MicrobiologyABSTRACT
Both sterile and non-sterile pharmaceutical products, which include antiseptics, have been recalled due to Burkholderia cepacia complex (BCC) contamination. Therefore, minimizing the frequency of outbreaks may be conducive to the development of a quick and sensitive approach that can distinguish between live and dead loads of BCC. We have assessed an exo probe-based recombinase polymerase amplification (RPA) with 10 µM propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in various concentrations of antiseptics (i.e., chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions) after 24 h. The optimized assay conducted using a set of primer-probes targeting gbpT was performed at 40 °C for 20 min and shows a detection limit of 10 pg/µL of genomic DNA from B. cenocepacia J2315, equivalent to 104 colony-forming units (CFU/mL). The specificity of a newly designed primer and probe was 80% (20 negatives out of 25). The readings for total cells (i.e., without PMAxx) from 200 µg/mL CHX using PMAxx-RPA exo assay was 310 relative fluorescence units (RFU), compared to 129 RFU with PMAxx (i.e., live cells). Furthermore, in 50-500 µg/mL BZK-treated cells, a difference in the detection rate was observed between the PMAxx-RPA exo assay in live cells (130.4-459.3 RFU) and total cells (207.82-684.5 RFU). This study shows that the PMAxx-RPA exo assay appears to be a valid tool for the simple, rapid and presumptive detection of live BCC cells in antiseptics, thereby ensuring the quality and safety of pharmaceutical products.
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.
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.
ABSTRACT
Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 µg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 µg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/µL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB-based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing.
