Optimizing bacterial DNA extraction in urine

Autoři: Matthew M. Munch aff001;  Laura C. Chambers aff002;  Lisa E. Manhart aff002;  Dan Domogala aff001;  Anthony Lopez aff001;  David N. Fredricks aff001;  Sujatha Srinivasan aff001
Působiště autorů: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America aff001;  Department of Epidemiology, University of Washington, Seattle, Washington, United states of America aff002;  Department of Global Health, University of Washington, Seattle, Washington, United states of America aff003;  Department of Medicine, University of Washington, Seattle, Washington, United States of America aff004;  Department of Microbiology, University of Washington, Seattle, Washington, United states of America aff005
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222962


Urine is an acceptable, non-invasive sample for investigating the human urogenital microbiota and for the diagnosis of sexually transmitted infections. However, low quantities of bacterial DNA and PCR inhibitors in urine may prevent efficient PCR amplification for molecular detection of bacteria. Furthermore, cold temperatures used to preserve DNA and bacteria in urine can promote precipitation of crystals that interfere with DNA extraction. Saline, Dulbecco’s Phosphate Buffered Saline, or Tris-EDTA buffer were added to urine from adult men to determine if crystal precipitation could be reversed without heating samples beyond ambient temperature. Total bacterial DNA concentrations and PCR inhibition were measured using quantitative PCR assays to compare DNA yields with and without buffer addition. Dissolution of crystals with Tris-EDTA prior to urine centrifugation was most effective in increasing bacterial DNA recovery and reducing PCR inhibition. DNA recovery using Tris-EDTA was further tested by spiking urine with DNA from bacterial isolates and median concentrations of Lactobacillus jensenii and Escherichia coli 16S rRNA gene copies were found to be higher in urine processed with Tris-EDTA. Maximizing bacterial DNA yield from urine may facilitate more accurate assessment of bacterial populations and increase detection of specific bacteria in the genital tract.

Klíčová slova:

Bacteria – DNA – DNA extraction – Oxalates – Polymerase chain reaction – Ribosomal RNA – Urine – Crystals


1. Nelson DE, Van Der Pol B, Dong Q, Revanna KV, Fan B, Easwaran S, et al. Characteristic male urine microbiomes associate with asymptomatic sexually transmitted infection. PLoS One. 2010;5:1–7. doi: 10.1371/journal.pone.0014116 21124791

2. Siddiqui H, Nederbragt AJ, Lagesen K, Jeansson SL, Jakobsen KS. Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons. BMC Microbiol. 2011;11:1–12.

3. Wolfe AJ, Toh E, Shibata N, Rong R, Kenton K, Fitzgerald M, et al. Evidence of uncultivated bacteria in the adult female bladder. J Clin Microbiol. 2012;50:1376–1383. doi: 10.1128/JCM.05852-11 22278835

4. Lewis DA, Brown R, Williams J, White P, Jacobson SK, Marchesi J, et al. The human urinary microbiome; bacterial DNA in voided urine of asymptomatic adults. Front Cell Infect Microbiol. 2013;3:41. doi: 10.3389/fcimb.2013.00041 23967406

5. Hilt EE, McKinley K, Pearce MM, Rosenfeld AB, Zilliox MJ, Mueller ER, et al. Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. J Clin Microbiol. 2014;52:871–876. doi: 10.1128/JCM.02876-13 24371246

6. Price TK, Dune T, Hilt EE, Thomas-White KJ, Kliethermes S, Brincat C, et al. The clinical urine culture: enhanced techniques improve detection of clinically relevant microorganisms. J Clin Microbiol. 2016;54:1216–1222. doi: 10.1128/JCM.00044-16 26962083

7. Thomas-White K, Brady M, Wolfe AJ, Mueller ER. The bladder is not sterile: History and current discoveries on the urinary microbiome. Curr Bladder Dysfunct Rep. 2016;11:18–24. doi: 10.1007/s11884-016-0345-8 27182288

8. Thomas-White K, Forster SC, Kumar N, Van Kuiken M, Putonti C, Stares MD, et al. Culturing of female bladder bacteria reveals an interconnected urogenital microbiota. Nat Commun. 2018;9:1557. doi: 10.1038/s41467-018-03968-5 29674608

9. Papp JR, Schachter J, Gaydos CA, Van Der Pol B. Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae—2014. MMWR. Recomm. Rep. 2014;63:1–19. Available from: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr6302a1.htm

10. Meyer T. Diagnostic procedures to detect Chlamydia trachomatis infections. Microorganisms. 2016;4:25. doi: 10.3390/microorganisms4030025 27681919

11. Napierala M, Munson E, Wenten D, Phipps P, Gremminger R, Schuknecht MK, et al. Detection of Mycoplasma genitalium from male primary urine specimens: an epidemiologic dichotomy with Trichomonas vaginalis. Diagn Microbiol Infect Dis. 2015;82:194–198. doi: 10.1016/j.diagmicrobio.2015.03.016 25934156

12. Van Der Pol B. Clinical and laboratory testing for Trichomonas vaginalis infection. J Clin Microbiol. 2016;54:7–12. doi: 10.1128/JCM.02025-15 26491181

13. Mckechnie ML, Hillman R, Couldwell D, Kong F, Freedman E, Wang H, et al. Simultaneous identification of 14 genital microorganisms in urine by use of a multiplex PCR-based reverse line blot assay. J Clin Microbiol. 2009;47:1871–1877. doi: 10.1128/JCM.00120-09 19357202

14. Schmidt K, Mwaigwisya S, Crossman LC, Doumith M, Munroe D, Pires C, et al. Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. J Antimicrob Chemother. 2017;72:104–114. doi: 10.1093/jac/dkw397 27667325

15. Nienhouse V, Gao X, Dong Q, Nelson DE, Toh E, McKinley K, et al. Interplay between bladder microbiota and urinary antimicrobial peptides: mechanisms for human urinary tract infection risk and symptom severity. PLoS One. 2014;9:e114185. doi: 10.1371/journal.pone.0114185 25486068

16. Horwitz D, McCue T, Mapes AC, Ajami NJ, Petrosino JF, Ramig RF, et al. Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference. J Infect. 2015;71:358–367. doi: 10.1016/j.jinf.2015.05.014 26048203

17. Liu F, Ling Z, Xiao Y, Yang Q, Zheng L, Jiang P, et al. Characterization of the urinary microbiota of elderly women and the effects of type 2 diabetes and urinary tract infections on the microbiota. Oncotarget. 2017;8:100678–100690. doi: 10.18632/oncotarget.21126 29246012

18. Moustafa A, Li W, Singh H, Moncera KJ, Torralba MG, Yu Y, et al. Microbial metagenome of urinary tract infection. Sci Rep. 2018;8:4333. doi: 10.1038/s41598-018-22660-8 29531289

19. Thomas-White KJ, Gao X, Lin H, Fok CS, Ghanayem K, Mueller ER, et al. Urinary microbes and postoperative urinary tract infection risk in urogynecologic surgical patients. Int Urogynecol J. 2018;29:1797–1805. doi: 10.1007/s00192-018-3767-3 30267143

20. Fok CS, Gao X, Lin H, Thomas-White KJ, Mueller ER, Wolfe AJ, et al. Urinary symptoms are associated with certain urinary microbes in urogynecologic surgical patients. Int Urogynecol J. 2018;29:1765–1771. doi: 10.1007/s00192-018-3732-1 30116843

21. Chen Z, Phan M-D, Bates LJ, Peters KM, Mukerjee C, Moore KH, et al. The urinary microbiome in patients with refractory urge incontinence and recurrent urinary tract infection. Int Urogynecol J. 2018;29:1775–1782. doi: 10.1007/s00192-018-3679-2 29946828

22. Pearce MM, Zilliox MJ, Thomas-White KJ, Richter HE, Nager CW, Visco AG, et al. The female urinary microbiota in urgency urinary incontinence. Am J Obstet Gynecol. 2015;213:347.e1–347.e11. doi: 10.1016/j.ajog.2015.07.009 26210757

23. Karstens L, Asquith M, Davin S, Stauffer P, Fair D, Gregory WT, et al. Does the urinary microbiome play a role in urgency urinary incontinence and its severity? Front Cell Infect Microbiol. 2016;6:78. doi: 10.3389/fcimb.2016.00078 27512653

24. Thomas-White KJ, Hilt EE, Fok C, Pearce MM, Mueller ER, Kliethermes S, et al. Incontinence medication response relates to the female urinary microbiota. Int Urogynecol J. 2016;27:723–733. doi: 10.1007/s00192-015-2847-x 26423260

25. Thomas-White KJ, Kliethermes S, Rickey L, Lukacz ES, Richter HE, Moalli P, et al. Evaluation of the urinary microbiota of women with uncomplicated stress urinary incontinence. Am J Obstet Gynecol. 2017;216:55.e1–55.e16. doi: 10.1016/j.ajog.2016.07.049 27498309

26. Komesu YM, Richter HE, Carper B, Dinwiddie DL, Lukacz ES, Siddiqui NY, et al. The urinary microbiome in women with mixed urinary incontinence compared to similarly aged controls. Int Urogynecol J. 2018;29:1785–1795. doi: 10.1007/s00192-018-3683-6 29909556

27. Wu P, Chen Y, Zhao J, Zhang G, Chen J, Wang J, et al. Urinary microbiome and psychological factors in women with overactive bladder. Front Cell Infect Microbiol. 2017;7:488. doi: 10.3389/fcimb.2017.00488 29230385

28. Siddiqui H, Lagesen K, Nederbragt AJ, Jeansson SL, Jakobsen KS. Alterations of microbiota in urine from women with interstitial cystitis. BMC Microbiol. 2012;12:205. doi: 10.1186/1471-2180-12-205 22974186

29. Abernethy MG, Rosenfeld A, White JR, Mueller MG, Lewicky-Gaupp C, Kenton K. Urinary microbiome and cytokine levels in women with interstitial cystitis. Obstet Gynecol. 2017;129:500–506. doi: 10.1097/AOG.0000000000001892 28178051

30. Gottschick C, Deng Z-L, Vital M, Masur C, Abels C, Pieper DH, et al. The urinary microbiota of men and women and its changes in women during bacterial vaginosis and antibiotic treatment. Microbiome. 2017;5:99. doi: 10.1186/s40168-017-0305-3 28807017

31. Frølund M, Wikström A, Lidbrink P, Abu Al-Soud W, Larsen N, Harder CB, et al. The bacterial microbiota in first-void urine from men with and without idiopathic urethritis. PLoS One. 2018;13:e0201380. doi: 10.1371/journal.pone.0201380 30052651

32. Wang H, Altemus J, Niazi F, Green H, Calhoun BC, Sturgis C, et al. Breast tissue, oral and urinary microbiomes in breast cancer. Oncotarget. 2017;8:88122–88138. doi: 10.18632/oncotarget.21490 29152146

33. Shrestha E, White JR, Yu S-H, Kulac I, Ertunc O, De Marzo AM, et al. Profiling the urinary microbiome in men with positive versus negative biopsies for prostate cancer. J Urol. 2018;199:161–171. doi: 10.1016/j.juro.2017.08.001 28797714

34. Wu P, Zhang G, Zhao J, Chen J, Chen Y, Huang W, et al. Profiling the urinary microbiota in male patients with bladder cancer in China. Front Cell Infect Microbiol. 2018;8:167. doi: 10.3389/fcimb.2018.00167 29904624

35. Bučević Popović V, Šitum M, Chow C-ET, Chan LS, Roje B, Terzić J. The urinary microbiome associated with bladder cancer. Sci Rep. 2018;8:12157. doi: 10.1038/s41598-018-29054-w 30108246

36. Alanee S, El-Zawahry A, Dynda D, Dabaja A, McVary K, Karr M, et al. A prospective study to examine the association of the urinary and fecal microbiota with prostate cancer diagnosis after transrectal biopsy of the prostate using 16sRNA gene analysis. Prostate. 2019;79:81–87. doi: 10.1002/pros.23713 30117171

37. Liu F, Ling Z, Xiao Y, Yang Q, Wang B, Zheng L, et al. Alterations of urinary microbiota in type 2 diabetes mellitus with hypertension and/or hyperlipidemia. Front Physiol. 2017;8:126. doi: 10.3389/fphys.2017.00126 28316574

38. Ling Z, Liu F, Shao L, Cheng Y, Li L. Dysbiosis of the urinary microbiota associated with urine levels of proinflammatory chemokine interleukin-8 in female type 2 diabetic patients. Front Immunol. 2017;8:1032. doi: 10.3389/fimmu.2017.01032 28943876

39. Kramer H, Kuffel G, Thomas-White K, Wolfe AJ, Vellanki K, Leehey DJ, et al. Diversity of the midstream urine microbiome in adults with chronic kidney disease. Int Urol Nephrol. 2018;50:1123–1130. doi: 10.1007/s11255-018-1860-7 29651696

40. Rani A, Ranjan R, McGee HS, Andropolis KE, Panchal DV, Hajjiri Z, et al. Urinary microbiome of kidney transplant patients reveals dysbiosis with potential for antibiotic resistance. Transl Res. 2017;181:59–70. doi: 10.1016/j.trsl.2016.08.008 27669488

41. Wu JF, Muthusamy A, Al-Ghalith GA, Knights D, Guo B, Wu B, et al. Urinary microbiome associated with chronic allograft dysfunction in kidney transplant recipients. Clin Transplant. 2018;32:e13436. doi: 10.1111/ctr.13436 30372560

42. Apoola A, Herrero-Diaz M, FitzHugh E, Rajakumar R, Fakis A, Oakden J. A randomised controlled trial to assess pain with urethral swabs. Sex Transm Infect. 2011;87:110–113. doi: 10.1136/sti.2010.042861 21131307

43. Dong Q, Nelson DE, Toh E, Diao L, Gao X, Fortenberry JD, et al. The microbial communities in male first catch urine are highly similar to those in paired urethral swab specimens. PLoS One. 2011;6:1–5. doi: 10.1371/journal.pone.0019709 21603636

44. Karstens L, Asquith M, Caruso V, Rosenbaum JT, Fair DA, Braun J, et al. Community profiling of the urinary microbiota: considerations for low-biomass samples. Nat Rev Urol. 2018;15:735–749. doi: 10.1038/s41585-018-0104-z 30315209

45. Khan G, Kangro HO, Coates PJ, Heath RB. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol. 1991;44:360–365. doi: 10.1136/jcp.44.5.360 1646235

46. Mahony J, Chong S, Jang D, Luinstra K, Faught M, Dalby D, et al. Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition. J Clin Microbiol. 1998;36:3122–3126. 9774550

47. Brunzel NA. Fundamentals of Urine and Body Fluid Analysis. 4th ed. St. Loius: Elsevier; 2018.

48. Fogazzi GB. Crystalluria: a neglected aspect of urinary sediment analysis. Nephrol Dial Transplant. 1996;11:379–387. doi: 10.1093/oxfordjournals.ndt.a027276 8671802

49. Ackerman AL, Anger JT, Khalique MU, Ackerman JE, Tang J, Kim J, et al. Optimization of DNA extraction from human urinary samples for mycobiome community profiling. PLoS One. 2019;14: e0210306. doi: 10.1371/journal.pone.0210306 31022216

50. Daudon M, Frochot V. Crystalluria. Clin Chem Lab Med. 2015;53:s1479–s1487. doi: 10.1515/cclm-2015-0860 26509782

51. Simerville J, Maxted W, Pahira J. Urinalysis: a comprehensive review. Am Fam Physician. 2005; 71:1153–1162. 15791892

52. Chambers LC, Morgan JL, Lowens MS, Robinson TS, Romano SS, Leipertz GL, et al. Cross-sectional study of urethral exposures at last sexual episode associated with non-gonococcal urethritis among STD clinic patients. Sex Transm Infect. 2018;sextrans-2018-053634. doi: 10.1136/sextrans-2018-053634 30181326

53. Saetun P, Semangoen T, Thongboonkerd V. Characterizations of urinary sediments precipitated after freezing and their effects on urinary protein and chemical analyses. Am J Physiol Renal Physiol. 2009;296:F1346–F1354. doi: 10.1152/ajprenal.90736.2008 19339629

54. Khot PD, Ko DL, Hackman RC, Fredricks DN. Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. BMC Infect Dis. 2008;8:1–13.

55. Srinivasan S, Hoffman NG, Morgan MT, Matsen FA, Fiedler TL, Hall RW, et al. Bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria. PLoS One. 2012;7:1–15. doi: 10.1371/journal.pone.0037818 22719852

56. Srinivasan S, Liu C, Mitchell CM, Fiedler TL, Thomas KK, Agnew KJ, et al. Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis. PLoS One. 2010;5:e10197. doi: 10.1371/journal.pone.0010197 20419168

57. Haber MH, Ward PCJ. Urine. In: McClatchey KD, editor. Clinical Laboratory Medicine. 2nd ed. Philadelphia: Lippincott Williams and Wilkins; 2002. pp. 533–534.

58. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. 2012;113:1014–1026. doi: 10.1111/j.1365-2672.2012.05384.x 22747964

Článek vyšel v časopise


2019 Číslo 9

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Zvyšte si kvalifikaci online z pohodlí domova

Ulcerative colitis_muž_břicho_střeva
Ulcerózní kolitida
nový kurz

Blokátory angiotenzinových receptorů (sartany)
Autoři: MUDr. Jiří Krupička, Ph.D.

Antiseptika a prevence ve stomatologii
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Citikolin v neuroprotekci a neuroregeneraci: od výzkumu do klinické praxe nejen očních lékařů
Autoři: MUDr. Petr Výborný, CSc., FEBO

Zánětlivá bolest zad a axiální spondylartritida – Diagnostika a referenční strategie
Autoři: MUDr. Monika Gregová, Ph.D., MUDr. Kristýna Bubová

Všechny kurzy
Kurzy Doporučená témata