Method for concentrating viable microorganisms for microbial load determination and eliminating uncertainty from matrix effects from urine and whole blood

Abstract

The ability to assess and eliminate the matrix effect in bioanalytical methods is critical for reproducibility, but sample preparation procedures necessary to address the matrix effect for microbiological methods could be significantly different if viable pathogens are required for downstream microbiological response analysis. A pure bacterial culture remains essential for virulence, antibiotic susceptibility, and phenotypic response studies in order to facilitate the understanding and treatment of caused diseases. Bacterial culture involves the collection, inoculation, incubation, growth, and detection of viable organisms while avoiding contamination throughout the entire process. The goal of this method is to concentrate viable pathogens directly from clinical specimens such as whole blood and urine while removing most interfering matrix components through pelleting in an enriched media, which is designed to facilitate the growth of clinically relevant microorganisms. Nonselective culture media with no inhibitors is used to permit the growth of most of the microorganisms present in the clinical samples studied. Most of the species implicated in clinical infections are mesophilic bacterial species, so the pelleting procedure is conducted at medium temperatures of 37°C to facilitate optimal growth.•Viable bacterial pelleting for phenotypic response analysis.•Concentration of bacteria by centrifugation and matrix component removal for direct-from-specimen molecular analysis.•Viable pathogen recovery directly from whole blood and urine.