Bacterial overgrowth affects urinary proteome analysis: recommendation for centrifugation, temperature, duration, and the use of preservatives during sample collection

Bacterial overgrowth is one of the major concerns in collection and storage of biofluids, particularly 24-h urine. However, there is no previous systematic analysis of effects of bacterial overgrowth on urinary proteome analysis, and necessity, type, and appropriate concentration of preservatives to prevent bacterial overgrowth in the urine remain unclear. We, therefore, performed such systematic evaluation. Pooled normal urine was either centrifuged at 1500 g (to remove cell debris) or uncentrifuged. The samples were then added with either sodium azide (NaN3) or boric acid with various concentrations, and kept at room temperature (RT) or at 4 degrees C. Bacterial overgrowth was determined by UV-visible spectrophotometry (lambda620 nm) and Gram staining. At both temperatures, centrifugation to remove cell debris could effectively delay the bacterial overgrowth. At RT, both centrifuged and uncentrifuged samples without any preservative had the detectable overgrowth of Gram-positive and Gram-negative cocci and bacilli as early as 12 and 8 h, respectively, whereas 0.1-1 mM NaN3 and 2-20 mM boric acid could delay bacterial overgrowth, which started at 16-20 h in the centrifuged urine and 12-16 h in the uncentrifuged urine. Greater delay (for at least 48 h) was achieved with 10 mM NaN3 and 200 mM boric acid. At 4 degrees C, no bacterial overgrowth was detected in all centrifuged samples. However, it was observed at 20 h in the uncentrifuged urine without preservative, and at 48 h for the uncentrifuged urine with 0.1 mM NaN3 or 2 mM boric acid. There was no bacterial overgrowth detectable in the uncentrifuged urine preserved with higher concentrations of NaN3 or boric acid. 2-DE showed obvious changes in the urinary proteome profile of the sample with bacterial contamination, and the bacterial proteins could be identified by MALDI-TOF MS. Our data suggest that the urine should be centrifuged to remove cell debris and kept at 4 degrees C, rather than at RT, during the collection interval prior to long-term storage in the freezer. Moreover, the addition of 200 mM boric acid or 10 mM NaN3 is highly recommended for the prevention of bacterial overgrowth in the urine.     

Analysis of differential proteomes in pathogenic and non‐pathogenic Leptospira: Potential pathogenic and virulence factors

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel‐based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non‐pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non‐pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.