Rapid separation of very low concentrations of bacteria from blood

Abstract

A rapid and accurate diagnosis of the species and antibiotic resistance of bacteria in septic blood is vital to increase survival rates of patients with bloodstream infections, particularly those with carbapenem-resistant enterobacteriaceae (CRE) infections. The extremely low levels in blood (1 to 100CFU/ml) make rapid diagnosis difficult. In this study, very low concentrations of bacteria (6 to 200CFU/ml) were separated from 7ml of whole blood using rapid sedimentation in a spinning hollow disk that separated plasma from red and white cells, leaving most of the bacteria suspended in the plasma. Following less than a minute of spinning, the disk was slowed, the plasma was recovered, and the bacteria were isolated by vacuum filtration. The filters were grown on nutrient plates to determine the number of bacteria recovered from the blood. Experiments were done without red blood cell (RBC) lysis and with RBC lysis in the recovered plasma. While there was scatter in the data from blood with low bacterial concentrations, the mean average recovery was 69%. The gender of the blood donor made no statistical difference in bacterial recovery. These results show that this rapid technique recovers a significant amount of bacteria from blood containing clinically relevant low levels of bacteria, producing the bacteria in minutes. These bacteria could subsequently be identified by molecular techniques to quickly identify the infectious organism and its resistance profile, thus greatly reducing the time needed to correctly diagnose and treat a blood infection.

Keywords: Bacterial infection; Blood sepsis; Carbapenem-resistant enterobacteriaceae; E. coli; Rapid diagnosis; Sedimentation.

Figure 1

Schematic of spinning blood in a hollow disk. a) Cross section of empty disk showing bowl, trough and weir. b) Upon spinning, blood is spun to the periphery of the disk. c) After 45 seconds at 3000 rpm, the RBCs and WBCs have sedimented quickly to form an outer layer, while most of the bacteria remains in the plasma layer. d) At conclusion of slow deceleration, the cells slide down into the trough while the plasma containing bacteria flows over the edge of the weir into the bowl where it is collected.

Figure 2

The % recovery as a function of CFU/ml in blood for 98 experiments. Triangles (▲) represent data from female blood that was quantitated without lysing the RBCs. The circles represent data from female (●) and male (○) blood with the RBCs lysed before filtering. The dashed lines indicate an estimated 95% prediction interval as a function of bacterial concentration.