High Sensitivity Detection of Bacillus Cereus in Plasma Samples.

Microbial contamination of blood and blood products remains a major concern in transfusion medicine. Detection of bacterial contamination has been traditionally carried out by inoculating the contaminated blood/blood products into a liquid or solid nutrient medium followed by 18–24 h incubation period to observe any bacterial colony formation (growth). Although this technique is highly sensitive and specific, the test requires at least 24 hours for accurate diagnosis, which is a limitation Nucleic acid-based and antigen-based assays that have been recently in use also have limitations either with regard to time, sensitivity or specificity. In the present study we utilized Bacillus cereus and bacteriolysin (PlyG) derived from its g-phage virus as a model system for high sensitivity detection of this B. cereus in plasma. Six overlapping synthetic peptides in the bacterial binding C-terminal region of PlyG were tested in the study. The length of the synthetic peptides ranged from 20 to 10-mer and were biotinylated for subsequent detection. Peptide binding to bacteria was detected using either Streptavidin-conjugated horse radish peroxidase (HRP) on a nitrocellulose membrane or by using streptavidin-conjugated fluorescent Q-dots (liquid nano crystals) and analyzing under a fluorescence microscope. Various dilutions of log-phase cultures of B. cereus were spiked into plasma samples and incubated with the peptides for 1 hour followed by detection using either the streptavidin-HRP or streptavidin-Q-dots. Results revealed that two out of six peptides bound strongly to Bacillus in both the assays, while the rest of the peptides were either non-binders or weakly-binding. The membrane-based assay demonstrated an assay sensitivity of 102 colony forming units (CFU)/ml whereas the Q dots-based fluorescence assay was able to detect even a single bacterium. Overall, our findings suggest that both these assays are rapid and sensitive and results could be achieved within 2–3 hours. Quantitation of the fluorescent signal by fluorometry in a 96-well plate format is in progress. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.