Cellulose-based preparative isoelectric focusing was used for preseparation and concentration of uropathogens Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Staphylococcus epidermidis, Candida albicans, and Candida parapsilosis in a urine sample containing a high concentration of human serum albumin. For the visibility of the colorless microbial zones in the separation medium, the microbial cells were labeled with red nonionogenic tenside (1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN). A very short incubation time, about 2 min, was sufficient for the adsorption of 0.001% (w/v) PAPAN onto the cell surface at the optimized conditions. As low as 103 cells of E. coli (pI 4.6) resuspended in 100 μL of urine sample and spiked with 0.1 mg mL-1 of human serum albumin (pI 4.8) were successfully preseparated and concentrated using this method. Because the pI values of the labeled microorganisms remained unchanged, the focused red zones of microbial cells were collected from the separation media and further analyzed by either capillary isoelectric focusing or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The viability of the cells extracted from the collected zones was also confirmed. The proposed method provides reliable, relatively fast, and cost-effective identification of uropathogens in urine specimens with a high level of albumin.

Institute of Analytical Chemistry of the CAS v v i Veveří 97 602 00 Brno Czech Republic

The Department of Microbiology Faculty of Medicine Masaryk University and St Anne's University Hospital Pekařská 53 602 00 Brno Czech Republic

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