Bacterial Overgrowth Affects Urinary Proteome Analysis - American

Bacterial Overgrowth Affects Urinary Proteome Analysis: Recommendation for Centrifugation, Temperature, Duration, and the Use of Preservatives during Sample Collection Visith Thongboonkerd* and Putita Saetun Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Received May 24, 2007

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 1500g (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 °C. Bacterial overgrowth was determined by UV-visible spectrophotometry (λ620 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 °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 °C, rather than at RT, during the collection interval prior to longterm 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. Keywords: Bacterial overgrowth • Boric acid • Preservatives • Proteome • Proteomics • Sodium azide • Urine • Urinary proteins

Introduction Recently, urinary proteomics has been widely applied to medical research with an ultimate goal of the discovery of novel biomarkers for earlier or more accurate diagnosis of human diseases, as well as for prediction and monitoring of therapeutic outcome.1,2 However, the urine is one of the most sophisticated biological samples to deal with, mainly because of the high degrees of inter-individual and intra-individual variabilities on urine volume, total protein concentration, and excreted levels of individual proteins.3-6 Moreover, bacterial overgrowth is one * Address correspondence to: Visith Thongboonkerd, MD, FRCPT, Medical Molecular Biology Unit, Office for Research and Development, 12th Floor Adulyadej Vikrom Building, Siriraj Hospital, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand. Phone/fax, +66-2-4184793; e-mails, [email protected] or [email protected]. 10.1021/pr070311+ CCC: $37.00

 2007 American Chemical Society

of the major concerns in urinary proteomics, particularly when the urine samples cannot be frozen immediately; that is, during 24-h collection, after the collection of one-void random urine but the sample cannot be processed immediately, thereby, has to be kept at room temperature or in a refrigerator for a while before it can be transferred into the freezer for the long-term storage. This gap interval may allow bacteria to grow until they can interfere with the urinary proteome analysis. Although bacterial overgrowth is a concern and is mentioned in a large number of references, the bacterial contamination during sample collection and storage of biological samples has not been well-demonstrated in proteomics studies. Nevertheless, several protocols for urinary proteome analysis have already included various kinds of preservatives to prevent bacterial overgrowth during urine collection,5,7-9 whereas some Journal of Proteome Research 2007, 6, 4173-4181

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Thongboonkerd and Saetun

overgrown bacteria, and the contaminated bacterial proteins could be identified in the urine samples using matrix-assited lase desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Time- and temperature-dependent bacterial overgrowth was observed, and difference in degree of bacterial overgrowth between the centrifuged and uncentrifuged urine samples was found. Effects of the preservatives on bacterial overgrowth were also examined.

Materials and Methods

Figure 1. Schematic summary of the study design in the present study.

others have not.6,10-12 Standardizations for urine collection and appropriate sample preparation methods are, therefore, crucially required. Previously, there was no systematic analysis on the necessity, type, and appropriate concentration of the preservatives in urinary proteome study. In the present study, we performed a systematic evaluation of the effects of bacterial overgrowth on the urinary proteome profile. Bacterial overgrowth was determined by UV-visible spectrophotometry (λ620 nm) and Gram staining. The urinary proteome profile using two-dimensional gel electrophoresis (2-DE) was obviously changed by the

The study design is summarized in Figure 1. Urine Collection and Preservation. First-morning, midstream urine samples were collected from 5 normal healthy individuals (3 females and 2 males; age ) 29.0 ( 4.0 years), who had no recent medication. All females had no menstrual cycle at the time of collection. Immediately after collection, the urine was pooled and divided into two sets; one was subjected to low-speed centrifugation (1500g for 15 min) to remove cell debris prior to next steps, while the other one was uncentrifuged. Both centrifuged and uncentrifuged urine samples were then added with either sodium azide (NaN3) (Merck KGaA; Damstadt, Germany) at various concentrations (0.1, 1, or 10 mM), boric acid (Merck Schuchardt OHG; Hohenbrunn, Germany) at various concentrations (2, 20, or 200 mM), or without any preservative. All individual samples were then divided into several tubes and kept at room temperature (RT) (∼28 °C) or at 4 °C for 0, 4, 8, 12, 16, 20, 24, and 48 h prior to the determination of bacterial overgrowth. Determination of Bacterial Overgrowth. The UV-visible spectrophotometer (Model 160A, Schimadzu; Kyoto, Japan) was employed to quantitatively analyze bacterial overgrowth, which usually causes progressive turbidity of the biofluids when the number of bacteria is progressively increased. The degree of

Figure 2. Gram staining of the overgrown bacteria observed in centrifuged (A and C) and uncentrifuged (B and D) urine samples kept at RT for 48 h without preservative. Gram-positive (blue) and Gram-negative (red) cocci (dot-like) and bacilli (rod-shaped) were observed. 4174

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Bacterial Overgrowth and Urinary Proteome

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Figure 3. Quantitative analysis of bacterial overgrowth in the centrifuged urine samples at different time points. The degree of turbidity, which reflects bacterial numbers, was measured using an UV-visible spectrophotometer at the wavelength of 620 nm (λ620).13,14 The data are reported as mean ( SD.

turbidity, which reflects bacterial numbers, was measured at the wavelength of 620 nm (λ620).13,14 The measurements were performed in 3 independent experiments, each of which was also triplicated (totally, 9 measurements for each sample). Differences in absorbance levels at λ620 nm were evaluated by ANOVA with Tukey’s post-hoc multiple comparisons. Pvalues 77 were considered statistically significant (P < 0.05). 4176


Thongboonkerd and Saetun Table 1. Summary of the Earliest Time Points, at Which Bacterial Overgrowth Was Detectablea the earliest time point, at which the bacterial overgrowth was observed condition

4 °C

Centrifuged Urine No preservative + NaN3 (0.1 mM) + NaN3 (1 mM) + NaN3 (10 mM) + Boric acid (2 mM) + Boric acid (20 mM) + Boric acid (200 mM) Uncentrifuged Urine No preservative 20 h + NaN3 (0.1 mM) 48 h + NaN3 (1 mM) + NaN3 (10 mM) + Boric acid (2 mM) 48 h + Boric acid (20 mM) + Boric acid (200 mM) -

RT

12 h 16 h 20 h 48 h 16 h 20 h 8h 12 h 12 h 48 h 12 h 16 h 48 h

a P < 0.05 compared to the baseline control at 0 h; n ) 3 independent experiments, each of which was also triplicated; totally 9 measurements for each sample.

Results and Discussion Two commonly used preservatives, NaN3 and boric acid, were applied in the present study to examine the inhibitory effects of the preservatives on bacterial overgrowth. The concentrations of NaN3 and boric acid used in the present study were based on their bacteriostatic and/or bactericidal concentrations, which had been used in several of previous urine studies.5,7,8,17-20 In addition to the use of varying concentrations of these preservatives, removal of cell debris, temperature, and time during the waiting or transit period of the sample (before transporting to the freezer) were also evaluated. Bacterial overgrowth was determined by UV-visible spectrophotometry and Gram stain. The absorbance at λ620 nm of the fresh normal centrifuged or uncentrifuged urine was comparable to that of dI water, which was used as the “blank” and set as “zero”. Because increasing number of bacteria in the urine caused progressive turbidity, the increase in the absorbance at λ620 nm was used as an indicator for bacterial overgrowth, which was subsequently confirmed by Gram staining. Figure 2 demonstrates numerous Gram-positive and Gramnegative cocci and bacilli in the centrifuged and uncentrifuged urine samples contaminated with bacteria. Our data were consistent with the findings reported in previous studies,18,19 which demonstrated that the overgrown bacteria commonly found in human urine included Enterobacteria (Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Proteus vulgaris, Morganella morganii, and Salmonella spp), other Gram-negative bacteria (Acinetobacter calcoaceticus, Acinetobacter lwoffii, and Pseudomonas aeruginosa), and Gram-positive cocci (Staphylococcus aureus, Staphylcoccus epidermidis, Staphyloccus saprophyticus, Streptococci group B, and Streptococci group D). Figure 3A shows the data obtained from quantitative analysis of bacterial overgrowth in the centrifuged urine kept at RT for 0, 4, 8, 12, 16, 20, 24, or 48 h. The sample without any preservative had the detectable bacterial overgrowth as early as 12 h, whereas 0.1-1 mM NaN3 and 2-20 mM boric acid could delay bacterial overgrowth, which started to be detected


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Figure 4. Quantitative analysis of bacterial overgrowth in the uncentrifuged urine samples at different time points. The degree of turbidity, which reflects bacterial numbers, was measured using an UV-visible spectrophotometer at the wavelength of 620 nm (λ620).13,14 The data are reported as mean ( SD.

at 16-20 h (Table 1). Greater delay (detectable only at 48 h) was achieved by 10 mM NaN3, whereas 200 mM boric acid completely abolished bacterial overgrowth for at least 48 h (Table 1). At 4 °C, there was no bacterial overgrowth observed in the centrifuged samples in the absence or presence of a preservative at any concentration (Figure 3B and Table 1). Figure 4A illustrates the data obtained from quantitative analysis of bacterial overgrowth in the uncentrifuged urine kept at RT for 0, 4, 8, 12, 16, 20, 24, or 48 h. The sample without any preservative had the detectable bacterial overgrowth as early as 8 h, whereas 0.1-1 mM NaN3 and 2-20 mM boric acid could delay bacterial overgrowth, which started to be detected at 1216 h (Table 1). With 10 mM NaN3 and 200 mM boric acid, the greater delay was observed (detectable only at 48 h) (Table 1).

At 4 °C, the bacterial overgrowth started 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 (Figure 4B and Table 1). There was no bacterial overgrowth detectable in the uncentrifuged urine preserved with higher concentrations of NaN3 or boric acid (Figure 4B and Table 1). When the centrifuged and uncentrifuged urine samples kept at 4 °C were compared, bacterial overgrowth was observed in some of the uncentrifuged samples (Figure 4B), whereas all of the centrifuged samples were still clear for at least 48 h (Figure 3B). Similarly, at RT, the uncentrifuged samples had bacterial overgrowth at earlier time points compared to those of the centrifuged urine. These data indicated that centrifugation to remove cell debris could effectively delay bacterial overgrowth Journal of Proteome Research • Vol. 6, No. 11, 2007 4177

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Figure 5. Changes in the urinary proteome profile by bacterial overgrowth in the centrifuged samples kept at RT without any preservative. The urine samples obtained from 5 healthy individuals were pooled and centrifuged at 1500g to remove cell debris. The samples were then divided into 8 aliquots of 1.6 mL and kept at RT for 0, 4, 8, 12, 16, 20, 24, or 48 h. Thereafter, proteins in these samples were concentrated using 75% ethanol precipitation method as described in more details in Materials and Methods. All the recovered proteins were then resolved with 2-DE (linear pH gradient of 3-10) and visualized with SYPRO Ruby stain. At the baseline (0 h), a total of 180 protein spots were detected using the predefined criteria of spot detection described in Materials and Methods. At 4 and 8 h, the 2-DE proteome profile remained unchanged. Starting from 12 h, the 2-DE proteome profile was progressively changing in relation to the incubation time. At 48 h, >500 newly presented protein spots were observed. The total protein recovered from 1.6 mL of these samples was increased from 60 µg at the baseline (0 h) to 245.52 µg at 48 h (approximately 4-fold increase). Some of these additional spots (labeled with numbers) were excised and identified by peptide mass fingerprinting using MALDI-TOF MS (see Table 2). Their quantitative intensity data are also shown in Figure 6. Table 2. Bacterial Proteins Identified by Peptide Mass Fingerprinting Using MALDI-TOF MS

spot no.

1 2 3 4 5 6 7 8 a

%cova

no. of matched masses (per queried masses)

theoretical pI

theoretical mass (kDa)

protein

NCBI ID

MOWSE score

Elongation factor EF-2 [Enterococcus faecalis V583] Ribosomal protein S1 [Serratia proteamaculans 568] Elongation factor Tu [Enterococcus faecalis V583] Enolase [Enterococcus faecalis V583] Elongation factor Tu [Escherichia coli CFT073] Glyceraldehyde-3-phosphate dehydrogenase [Enterobacter aerogenes] Glyceraldehyde-3-phosphate dehydrogenase [Klebsiella pneumoniae subsp. pneumoniae MGH 78578] Outer membrane protein X precursor [Enterobacter cloacae]

gi|29374846 gi|118068082 gi|29374847 gi|29376483 gi|26249935 gi|120692

290 177 253 240 239 90

51 42 70 70 77 44

24/42 18/43 20/37 19/35 23/55 8/26

4.80 4.94 4.73 4.56 5.25 6.27

76.63 61.48 43.42 46.48 44.99 31.60

gi|152969751

105

33

10/30

6.61

35.62

gi|129164

127

69

10/40

7.74

18.70

%Cov (sequence coverage) ) [number of the identified residues/total number of amino acid residues in the protein sequence] × 100%.

and implicated that cell debris might serve as the useful nutrient resource for bacteria to grow and, thus, could accelerate bacterial overgrowth. In parallel, we also performed 2-DE to evaluate changes in the urinary proteome profile affected by bacterial overgrowth. The 2-DE proteome profiles of the centrifuged urine samples without preservative and with 200 mM boric acid kept at RT for 0, 4, 8, 12, 16, 20, 24, and 48 h were thus evaluated. Because these samples were from the same urine pool, an equal volume of 1.6 mL was used for all the samples to examine changes in 4178


the urinary proteome profile caused by bacterial overgrowth. At the final resuspension step after dialysis (to remove salts and interfering compounds) and lyophilization, a fixed volume (150 µL) of the rehydration buffer was used. With this fixed volume, there were no unsolubilized particles observed. Figure 5 shows 2-DE proteome profiles of the centrifuged urine samples that were collected without preservative and kept at RT for 0-48 h (this set of samples showed bacterial overgrowth, which was detectable by UV-visible spectrophotometry as early as 12 h; see Table 1). At the baseline (0 h), a


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Figure 6. Quantitative intensity data of the newly expressed protein spots, which were selected for subsequent identification by peptide mass fingerprinting using MALDI-TOF MS. Their identities are shown in Table 2. The numbers indicated in this figure correspond to those reported in Table 2 and labeled in Figure 5.

Figure 7. Successful prevention of bacterial overgrowth using 200 mM boric acid. The urine samples obtained from 5 healthy individuals were pooled and centrifuged at 1500g to remove cell debris, and 200 mM boric acid was added. The samples were then divided into 8 aliquots of 1.6 mL and kept at RT for 0, 4, 8, 12, 16, 20, 24, or 48 h. Thereafter, proteins in these samples were concentrated using 75% ethanol precipitation method as described in more details in Materials and Methods. All the recovered proteins were then resolved with 2-DE (linear pH gradient of 3-10) and visualized with SYPRO Ruby stain. For at least 48 h, the urinary proteome profile remained unchanged, and no newly presented protein spots were observed in this set of the samples.

total of 180 protein spots was detected using the predefined criteria of spot detection described in Materials and Methods, and the 2-DE proteome profile (pattern of protein spots) was consistent with the 2-D proteome maps of the normal human urine described previously.3,4,7,21 At 4 and 8 h, the 2-DE proteome profile remained unchanged. Starting from 12 h, the 2-DE proteome profile was progressively changed in relation to the incubation time. At 48 h, >500 newly presented protein spots were observed in the 2-DE gel. The total protein recovered from 1.6 mL of these samples (in another parallel experiment) was increased from 60 µg at the baseline (0 h) to 245.52 µg at 48 h (approximately 4-fold increase).

These additional spots and the increased amount of total protein were most likely originated from the overgrown bacteria. To confirm this, some of the newly presented protein spots were then excised and subjected to the identification by peptide mass fingerprinting using MALDI-TOF MS. The data reported in Table 2 clearly confirmed that these additional or contaminated proteins were originated from the overgrown bacteria. Their quantitative intensity data shown in Figure 6 were consistent with the UV-visible spectrophotometric data illustrated in Figures 3 and 4 and in Table 1, all of which indicated that the bacterial overgrowth in the centrifuged urine kept at RT without any preservative was detectable as early as Journal of Proteome Research • Vol. 6, No. 11, 2007 4179

research articles 12 h. Subsequently, the bacterial overgrowth was clearly confirmed in these samples by Gram staining (Figure 2A,C). In contrast, Figure 7 clearly shows that the addition of 200 mM boric acid into the centrifuged urine kept at RT could effectively prevent bacterial overgrowth for at least 48 h, as there were no newly presented protein spots observed in this set of the samples. To the best of our knowledge, this data set is the first that provides the direct evidence of effects of bacterial overgrowth on the urinary proteome profile. Prevention of bacterial overgrowth in the urine is beneficial for urinary proteome analysis regardless of proteomic technologies used. Our present study has addressed an important issue for the “sample collection and storage” prior to proteome analysis. Although we used 2-DE as a model for demonstrating effects of bacterial overgrowth on the urinary proteome profile, the data reported herein can be also beneficial for any other proteomic technologies applied to urinary proteome analysis (i.e., LC-MS/MS, SELDI-TOF MS, CE-TOF MS, microarrays).2,22 Moreover, these data will be also applicable to conventional clinical chemistry tests. One might argue that a preservative may not be required if the urine sample can be frozen immediately after the collection for the long-term storage (which is absolutely right). However, the unexpected delay in transportation of urine samples in several hospitals often affects the duration between sample collection and long-term storage in the freezer. The specimens can become overgrown with organisms during this gap interval, in which most of the specimens are kept at RT or in a refrigerator prior to transporting to the laboratory. Bacterial overgrowth is also a major problem for 24-h collection of the urine from both outpatients and hospitalized cases. Because overgrown bacteria can produce a large number of bacterial proteins, which definitely interfere with urinary proteome analysis, prevention of bacterial overgrowth using preservatives is, thus, crucial for the urinary proteome study. It should be emphasized that our data were obtained from the normal urine collected from healthy individuals. The quantitative data of bacterial overgrowth might not represent those in the diseased urine obtained from patients with urinary tract infections, glomerular disorders, autoimmune diseases, urological stones, and so forth that may have a higher risk or greater degree of the bacterial growth. Additionally, this set of the data might not be applicable to the sterile urine obtained carefully from a percutaneous cystostomy or from a surgical intervention. In summary, our data clearly indicate that bacterial overgrowth is a major obstacle in urinary proteomics. Therefore, its prevention is crucial for the urinary proteome analysis. On the basis of the data reported herein, our recommendations for the prevention of bacterial overgrowth in the urine are as follows: • Cell debris should be removed from the urine by low-speed centrifugation (1,000-1500g for 10-20 min) prior to the storage. • During the waiting or transit period, the urine should be kept at 4 °C, not at RT. • The urine sample should not be kept at RT for longer than 8 h without any preservative. • For the collection of one-void random urine, 2-20 mM boric acid or 0.1-1 mM NaN3, which can delay the bacterial overgrowth for 12-20 h at RT, should be added. • For 24-h urine collection, the addition of 200 mM boric acid or 10 mM NaN3 is highly recommended. 4180



Abbreviations: 2-DE, two-dimensional gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate; dI water, 18 MΩ·cm water; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; MALDITOF MS, matrix-assisted laser desorption/ionization time-offlight mass spectrometry; NaN3, sodium azide; RT, room temperature.

Acknowledgment. We are grateful to Wararat Chiangjong for her technical assistance. This study was supported by Siriraj Grant for Research and Development, The Thailand Research Fund, Commission on Higher Education, Mahidol University, the National Research Council of Thailand, and the National Center for Genetic Engineering and Biotechnology. References (1) Thongboonkerd, V.; Malasit, P. Renal and urinary proteomics: Current applications and challenges. Proteomics 2005, 5, 10331042. (2) Thongboonkerd, V. Recent progress in urinary proteomics. Proteomics Clin. Appl. 2007, 1, 780-791. (3) Oh, J.; Pyo, J. H.; Jo, E. H.; Hwang, S. I.; Kang, S. C.; Jung, J. H.; Park, E. K.; Kim, S. Y.; Choi, J. Y.; Lim, J. Establishment of a nearstandard two-dimensional human urine proteomic map. Proteomics 2004, 4, 3485-3497. (4) Thongboonkerd, V.; Chutipongtanate, S.; Kanlaya, R. Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: quantity, quality, and variability. J. Proteome Res. 2006, 5, 183-191. (5) Zhou, H.; Yuen, P. S.; Pisitkun, T.; Gonzales, P. A.; Yasuda, H.; Dear, J. W.; Gross, P.; Knepper, M. A.; Star, R. A. Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int. 2006, 69, 14711476. (6) Khan, A.; Packer, N. H. Simple urinary sample preparation for proteomic analysis. J. Proteome Res. 2006, 5, 2824-2838. (7) Thongboonkerd, V.; McLeish, K. R.; Arthur, J. M.; Klein, J. B. Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation. Kidney Int. 2002, 62, 1461-1469. (8) Pisitkun, T.; Shen, R. F.; Knepper, M. A. Identification and proteomic profiling of exosomes in human urine. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 13368-13373. (9) Sun, W.; Li, F.; Wu, S.; Wang, X.; Zheng, D.; Wang, J.; Gao, Y. Human urine proteome analysis by three separation approaches. Proteomics 2005, 5, 4994-5001. (10) Castagna, A.; Cecconi, D.; Sennels, L.; Rappsilber, J.; Guerrier, L.; Fortis, F.; Boschetti, E.; Lomas, L.; Righetti, P. G. Exploring the hidden human urinary proteome via ligand library beads. J. Proteome Res. 2005, 4, 1917-1930. (11) Park, M. R.; Wang, E. H.; Jin, D. C.; Cha, J. H.; Lee, K. H.; Yang, C. W.; Kang, C. S.; Choi, Y. J. Establishment of a 2-D human urinary proteomic map in IgA nephropathy. Proteomics 2006, 6, 1066-1076. (12) Adachi, J.; Kumar, C.; Zhang, Y.; Olsen, J. V.; Mann, M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. GenomeBiology 2006, 7, R80. (13) Carson, C. F.; Mee, B. J.; Riley, T. V. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob. Agents Chemother. 2002, 46, 1914-1920. (14) Zhao, L.; Montville, T. J.; Schaffner, D. W. Time-to-detection, percent-growth-positive and maximum growth rate models for Clostridium botulinum 56A at multiple temperatures. Int. J. Food Microbiol. 2002, 77, 187-197. (15) Thongboonkerd, V.; Luengpailin, J.; Cao, J.; Pierce, W. M.; Cai, J.; Klein, J. B.; Doyle, R. J. Fluoride exposure attenuates expression of Streptococcus pyogenes virulence factors. J. Biol. Chem. 2002, 277, 16599-16605. (16) Thongboonkerd, V.; Gozal, E.; Sachleben, L. R.; Arthur, J. M.; Pierce, W. M.; Cai, J.; Chao, J.; Bader, M.; Pesquero, J. B.; Gozal, D.; Klein, J. B. Proteomic analysis reveals alterations in the renal kallikrein pathway during hypoxia-induced hypertension. J. Biol. Chem. 2002, 277, 34708-34716.

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Bacterial Overgrowth and Urinary Proteome (17) Nickander, K. K.; Shanholtzer, C. J.; Peterson, L. R. Urine culture transport tubes: effect of sample volume on bacterial toxicity of the preservative. J. Clin. Microbiol. 1982, 15, 593-595. (18) Meers, P. D.; Chow, C. K. Bacteriostatic and bactericidal actions of boric acid against bacteria and fungi commonly found in urine. J. Clin. Pathol. 1990, 43, 484-487. (19) Gillespie, T.; Fewster, J.; Masterton, R. G. The effect of specimen processing delay on borate urine preservation. J. Clin. Pathol. 1999, 52, 95-98. (20) Hoppin, J. A.; Ulmer, R.; Calafat, A. M.; Barr, D. B.; Baker, S. V.; Meltzer, H. M.; Ronningen, K. S. Impact of urine preservation methods and duration of storage on measured levels of environmental contaminants. J. Exposure Sci. Environ. Epidemiol. 2006, 16, 39-48.

(21) Pieper, R.; Gatlin, C. L.; McGrath, A. M.; Makusky, A. J.; Mondal, M.; Seonarain, M.; Field, E.; Schatz, C. R.; Estock, M. A.; Ahmed, N.; Anderson, N. G.; Steiner, S. Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 2004, 4, 1159-1174. (22) Thongboonkerd, V. Practical points in urinary proteomics. J. Proteome Res. 2007, 6, 3881-3890.

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