Bacterial Overgrowth Affects Urinary Proteome Analysis:  Recommendation for Centrifugation, Temperature, Duration, and the Use of Preservatives during Sample Collection

Abstract

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.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

“…Prior to the use of MALDI-TOF MS for the diagnosis of urinary tract infections (UTIs), the presence of blood and bacteria in the urine was known to interfere with urinary proteomic analysis, specifically altering key peptide mass signals in the sample (299). Additional studies similarly suggested that bacterial overgrowth of the urine could hamper proteomic analysis and recommended that samples be centrifuged and stored immediately at 4°C and have boric acid or NaN 3 added to prevent bacteria from overgrowing (300). It remains to be seen which specimen handling conditions are necessary for optimal identification of bacteria in urinary samples, as higher bacterial burdens within the specimen could potentially simplify the detection of the bac-teria in urine samples at the expense of the rest of the urinary proteome.…”

MALDI-TOF MS: Its Application in the Clinical Laboratory and a Paradigm Shift in Clinical Microbiology

“…Prior to the use of MALDI-TOF MS for the diagnosis of UTIs, the presence of blood and bacteria in the urine was reported to interfere with urinary proteomic analysis, specifically altering key peptide-mass signals in the sample [24,25]. An additional report similarly suggested that bacterial overgrowth of the urine could hamper proteomic analysis, and recommended that samples be immediately centrifuged and stored at 4˚C, and have boric acid or sodium azide added to prevent bacteria from overgrowing [26]. Which specimen handling conditions are necessary for optimal identification of bacteria in urinary samples remains to be seen, as a high bacterial burden within urine samples could potentially simplify the detection of the bacteria at the expense of the rest of the urinary proteome.…”

Human Urine Proteome: A Powerful Source for Clinical Research

“…During the storage, urinary samples might have bacterial overgrowth, which could change the urinary proteome [19]. Therefore, preservatives were recommended to prevent the bacterial overgrowth after collection [19].…”