(311c) Separation of Bacteria from Blood Components

Authors: 
Pitt, W. G., Brigham Young University
Alizadeh, M., Brigham Young University
Husseini, G. A., American University of Sharjah

Blood bacteremia, or a bacteria infection in the blood stream, is of major concern in terms of mortality and morbidity, particularly when antibiotic resistant bacteria are involved. Persons presenting bacteremia symptoms are often given standard doses of general antibiotics and a blood sample is taken to culture the bacteria for identification.  The problem is that culturing and identifying the pathogen in order to administer the correct antibiotic response usually requires 24 to 48 hours, during which time the patient could become severely sick if the particular pathogen is resistant to the general antibiotic already administered. 

Our overall goal is to develop methods that are much faster, even as fast as one hour, that can identify antibiotic resistant bacteria from a person’s blood.  This implies that there is no time to culture the bacteria in the blood to obtain sufficient numbers of bacteria to identify resistance by current conventional means.  We are developing methods to quickly identify the bacteria in the blood using molecular mechanisms such as direct DNA or RNA identification without PCR amplification of certain gene sequences and without culture amplification of the bacteria.  The first step in this process is to separate the bacteria from the blood components that would interfere with the subsequent assays.  The means separating the bacteria from the red blood cells (RBCs), white blood cells (WBCs) and platelets (PLTs). The target time is 10 minutes or less for separation, after which the recovered bacteria will be lysed and their DNA analyzed for identification within 50 minutes.

This submission presents our successes and failures in using porous hollow fibers, flat membrane filters and centrifugal processes for separation. 

While the use of porous hollow fibers with 3-5 um pores is good in theory, the application is difficult because hollow fibers with uniform cylindrical pores are not commercially available.  Porous hollow fibers with elliptical pores of 5 um size are available, but these trap bacteria (not pass bacteria) because of the pore shape. Pores with tortuous pathways also trap bacteria that would otherwise pass through a cylindrical pore.

Flat membranes with uniform pores are commercially available, and have been used in this work with bovine blood.  However, the bacteria recovery is low, and the area needed to prevent filter cake build-up is so large that a small disposable device that could be used in a hospital becomes impractical in terms of size and expense.

Centrifugal separation can easily separate RBCs (~8 um) and WBCs (~15 um) from platelets (~3 um) and bacteria (0.5 to 2 um) because of the large size of the cells. The dynamic sedimentation velocity goes as the diameter squared, and red cells are relatively dense because of their hemoglobin content. In these preliminary studies we have used bovine blood spiked with E. coli. We have had success in centrifugal separation with high bacterial recovery as only a small fraction of bacteria gets trapped in packed cells.  We have developed and will present some unique centrifugation procedures that increase the efficiency or separation of cells from bacteria.