1887

Abstract

Studies of bacterial transcriptomics during bloodstream infections are limited to-date because unbiased extraction of bacterial mRNA from whole blood in sufficient quantity and quality has proved challenging. Problems include the high excess of human cells, the presence of PCR inhibitors and the short intrinsic half-life of bacterial mRNA. This study aims to provide a framework for the choice of the most suitable sample preparation method.

cells were spiked into human whole blood and the bacterial gene expression was stabilized with RNAprotect either immediately or after lysis of the red blood cells with Triton X-100, saponin, ammonium chloride or the commercial MolYsis buffer CM. RNA yield, purity and integrity were assessed by absorbance measurements at 260 and 280 nm, real-time PCR and capillary electrophoresis.

For low cell numbers, the best mRNA yields were obtained by adding the commercial RNAprotect reagent directly to the sample without prior lyses of the human blood cells. Using this protocol, significant amounts of human RNA were co-purified, however, this had a beneficial impact on the yields of bacterial mRNA. Among the tested lysis agents, Triton X-100 was the most effective and reduced the human RNA background by three to four orders of magnitude.

For most applications, lysis of the human blood cells is not required. However, co-purified human RNA may interfere with some downstream processes such as RNA sequencing. In this case, blood cell lysis with Triton X-100 is desirable.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000439
2017-03-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/66/3/301.html?itemId=/content/journal/jmm/10.1099/jmm.0.000439&mimeType=html&fmt=ahah

References

  1. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003; 348:1546–1554 [View Article][PubMed]
    [Google Scholar]
  2. Artero A, Zaragoza R, Camarena JJ, Sancho S, González R et al. Prognostic factors of mortality in patients with community-acquired bloodstream infection with severe sepsis and septic shock. J Crit Care 2010; 25:276–281 [View Article][PubMed]
    [Google Scholar]
  3. Lark RL, Saint S, Chenoweth C, Zemencuk JK, Lipsky BA et al. Four-year prospective evaluation of community-acquired bacteremia: epidemiology, microbiology, and patient outcome. Diagn Microbiol Infect Dis 2001; 41:15–22 [View Article][PubMed]
    [Google Scholar]
  4. Macfarlane DE, Dahle CE. Isolating RNA from whole blood—the dawn of RNA-based diagnosis?. Nature 1993; 362:186–188 [View Article][PubMed]
    [Google Scholar]
  5. Graham MR, Virtaneva K, Porcella SF, Barry WT, Gowen BB et al. Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies. Am J Pathol 2005; 166:455–465 [View Article][PubMed]
    [Google Scholar]
  6. Barczak AK, Gomez JE, Kaufmann BB, Hinson ER, Cosimi L et al. RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities. Proc Natl Acad Sci USA 2012; 109:6217–6222 [View Article][PubMed]
    [Google Scholar]
  7. Wilson J, Elgohari S, Livermore DM, Cookson B, Johnson A et al. Trends among pathogens reported as causing bacteraemia in England, 2004-2008. Clin Microbiol Infect 2011; 17:451–458 [View Article][PubMed]
    [Google Scholar]
  8. Opota O, Croxatto A, Prod'hom G, Greub G. Blood culture-based diagnosis of bacteraemia: state of the art. Clin Microbiol Infect 2015; 21:313–322 [View Article][PubMed]
    [Google Scholar]
  9. Bhatty M, Fan R, Muir WM, Pruett SB, Nanduri B. Transcriptomic analysis of peritoneal cells in a mouse model of sepsis: confirmatory and novel results in early and late sepsis. BMC Genomics 2012; 13:509 [View Article][PubMed]
    [Google Scholar]
  10. Smith SN, Hagan EC, Lane MC, Mobley HLT. Dissemination and systemic colonization of uropathogenic Escherichia coli in a murine model of bacteremia. MBio 2010; 1:e00262-10 [View Article][PubMed]
    [Google Scholar]
  11. Skorup P, Maudsdotter L, Lipcsey M, Castegren M, Larsson A et al. Beneficial antimicrobial effect of the addition of an aminoglycoside to a β-lactam antibiotic in an E. coli porcine intensive care severe sepsis model. PLoS One 2014; 9:e90441 [View Article][PubMed]
    [Google Scholar]
  12. Matsuda K, Tsuji H, Asahara T, Kado Y, Nomoto K. Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR. Appl Environ Microbiol 2007; 73:32–39 [View Article][PubMed]
    [Google Scholar]
  13. Fujimori M, Hisata K, Nagata S, Matsunaga N, Komatsu M et al. Efficacy of bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR for detecting neonatal sepsis: a case control study. BMC Pediatr 2010; 10:53 [View Article][PubMed]
    [Google Scholar]
  14. Sakaguchi S, Saito M, Tsuji H, Asahara T, Takata O et al. Bacterial rRNA-targeted reverse transcription-PCR used to identify pathogens responsible for fever with neutropenia. J Clin Microbiol 2010; 48:1624–1628 [View Article][PubMed]
    [Google Scholar]
  15. Bremer H, Dennis PP. Modulation of chemical composition and other parameters of the cell at different exponential growth rates. EcoSal Plus 2008; 3:765–777 [View Article][PubMed]
    [Google Scholar]
  16. Arfvidsson C, Wahlund KG. Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field–flow fractionation. Anal Biochem 2003; 313:76–85 [View Article][PubMed]
    [Google Scholar]
  17. Akane A, Matsubara K, Nakamura H, Takahashi S, Kimura K. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci 1994; 39:362–372 [View Article][PubMed]
    [Google Scholar]
  18. Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I. Effects of heparin on polymerase chain reaction for blood white cells. J Clin Lab Anal 1999; 13:133–140 [View Article][PubMed]
    [Google Scholar]
  19. Morata P, Queipo-Ortuño MI, de Dios Colmenero J. Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J Clin Microbiol 1998; 36:2443–2446[PubMed]
    [Google Scholar]
  20. Al-Soud WA, Jönsson LJ, Râdström P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 2000; 38:345–350[PubMed]
    [Google Scholar]
  21. Song Y, Giske CG, Gille-Johnson P, Emanuelsson O, Lundeberg J et al. Nuclease-assisted suppression of human DNA background in sepsis. PLoS One 2014; 9:e103610 [View Article][PubMed]
    [Google Scholar]
  22. Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol 2009; 2009:1–10 [View Article]
    [Google Scholar]
  23. Bernstein JA, Khodursky AB, Lin PH, Lin-Chao S, Cohen SN. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc Natl Acad Sci USA 2002; 99:9697–9702 [View Article][PubMed]
    [Google Scholar]
  24. Hambraeus G, von Wachenfeldt C, Hederstedt L. Genome-wide survey of mRNA half-lives in Bacillus subtilis identifies extremely stable mRNAs. Mol Genet Genomics 2003; 269:706–714 [View Article][PubMed]
    [Google Scholar]
  25. Rogacs A, Qu Y, Santiago JG. Bacterial RNA extraction and purification from whole human blood using isotachophoresis. Anal Chem 2012; 84:5858–5863 [View Article][PubMed]
    [Google Scholar]
  26. Hedman ÅK, Hedman AK, Li MS, Langford PR, Kroll JS. Transcriptional profiling of serogroup B Neisseria meningitidis growing in human blood: an approach to vaccine antigen discovery. PLoS One 2012; 7:e39718 [View Article][PubMed]
    [Google Scholar]
  27. Del Tordello E, Bottini S, Muzzi A, Serruto D. Analysis of the regulated transcriptome of Neisseria meningitidis in human blood using a tiling array. J Bacteriol 2012; 194:6217–6232 [View Article][PubMed]
    [Google Scholar]
  28. Echenique-Rivera H, Muzzi A, Del Tordello E, Seib KL, Francois P et al. Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival. PLoS Pathog 2011; 7:e1002027 [View Article][PubMed]
    [Google Scholar]
  29. Mereghetti L, Sitkiewicz I, Green NM, Musser JM. Extensive adaptive changes occur in the transcriptome of Streptococcus agalactiae (group B Streptococcus) in response to incubation with human blood. PLoS One 2008; 3:e3143 [View Article][PubMed]
    [Google Scholar]
  30. Orihuela CJ, Radin JN, Sublett JE, Gao G, Kaushal D et al. Microarray analysis of pneumococcal gene expression during invasive disease. Infect Immun 2004; 72:5582–5596 [View Article][PubMed]
    [Google Scholar]
  31. Shortman K, Williams N, Adams P. The separation of different cell classes from lymphoid organs. V. Simple procedures for the removal of cell debris. damaged cells and erythroid cells from lymphoid cell suspensions. J Immunol Methods 1972; 1:273–287 [View Article][PubMed]
    [Google Scholar]
  32. Mccann CD, Jordan JA. Evaluation of MolYsis Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing. J Microbiol Methods 2014; 99:1–7 [View Article][PubMed]
    [Google Scholar]
  33. Hansen WLJ, Bruggeman CA, Wolffs PFG. Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood; 2009; 472629–2631
  34. Bangham AD, Horne RW, Glauert AM, Dingle JT, Lucy JA. Action of saponin on biological cell membranes. Nature 1962; 196:952–953 [View Article][PubMed]
    [Google Scholar]
  35. Chen JH, Ho PL, Kwan GS, She KK, Siu GK et al. Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. J Clin Microbiol 2013; 51:1733–1739 [View Article][PubMed]
    [Google Scholar]
  36. Meex C, Neuville F, Descy J, Huynen P, Hayette MP et al. Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction. J Med Microbiol 2012; 61:1511–1516 [View Article][PubMed]
    [Google Scholar]
  37. Biocartis SA Selective lysis of cells by ionic surfactants WIPO, WO/2012/168003 2012
  38. Zelenin S, Hansson J, Ardabili S, Ramachandraiah H, Brismar H et al. Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics. Biotechnol Lett 2015; 37:825–830 [View Article][PubMed]
    [Google Scholar]
  39. QIAGEN ltd RNAprotect bacteria reagent MSDS 2015
  40. Rainen L, Oelmueller U, Jurgensen S, Wyrich R, Ballas C et al. Stabilization of mRNA expression in whole blood samples. Clin Chem 2002; 48:1883–1890[PubMed]
    [Google Scholar]
  41. Welch RA, Burland V, Plunkett G, Redford P, Roesch P et al. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc Natl Acad Sci USA 2002; 99:17020–17024 [View Article][PubMed]
    [Google Scholar]
  42. Luo C, Hu GQ, Zhu H. Genome reannotation of Escherichia coli CFT073 with new insights into virulence. BMC Genomics 2009; 10:552 [View Article][PubMed]
    [Google Scholar]
  43. Chung HJ, Bang W, Drake MA. Stress response of Escherichia coli. Compr Rev Food Sci Food Saf 2006; 5:52–64 [View Article]
    [Google Scholar]
  44. Danese PN, Silhavy TJ. CpxP, a stress-combative member of the Cpx regulon. J Bacteriol 1998; 180:831–839[PubMed]
    [Google Scholar]
  45. Darwin AJ. The phage-shock-protein response. Mol Microbiol 2005; 57:621–628 [View Article][PubMed]
    [Google Scholar]
  46. Maurer LM, Yohannes E, Bondurant SS, Radmacher M, Slonczewski JL. pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12. J Bacteriol 2005; 187:304–319 [View Article][PubMed]
    [Google Scholar]
  47. Weiner L, Brissette JL, Model P. Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on sigma 54 and modulated by positive and negative feedback mechanisms. Genes Dev 1991; 5:1912–1923 [View Article][PubMed]
    [Google Scholar]
  48. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001; 25:402–408 [View Article][PubMed]
    [Google Scholar]
  49. Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res 2005; 33:e56 [View Article][PubMed]
    [Google Scholar]
  50. Di Cello F, Xie Y, Paul-Satyaseela M, Kim KS. Approaches to bacterial RNA isolation and purification for microarray analysis of Escherichia coli K1 interaction with human brain microvascular endothelial cells. J Clin Microbiol 2005; 43:4197–4199 [View Article][PubMed]
    [Google Scholar]
  51. La MV, Raoult D, Renesto P. Regulation of whole bacterial pathogen transcription within infected hosts. FEMS Microbiol Rev 2008; 32:440–460 [View Article][PubMed]
    [Google Scholar]
  52. Rienksma RA, Suarez-Diez M, Mollenkopf HJ, Dolganov GM, Dorhoi A et al. Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing. BMC Genomics 2015; 16:34 [View Article][PubMed]
    [Google Scholar]
  53. Fleige S, Walf V, Huch S, Prgomet C, Sehm J et al. Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett 2006; 28:1601–1613 [View Article][PubMed]
    [Google Scholar]
  54. Molzym Pathogen DNA extraction and PCR analysis, version 3.1. UMD - Univers 2014
  55. Brunck MEG, Andersen SB, Timmins NE, Osborne GW, Nielsen LK. Absolute counting of neutrophils in whole blood using flow cytometry. Cytometry A 2014; 85:1057–1064 [View Article]
    [Google Scholar]
  56. Boyd MA, Tennant SM, Melendez JH, Toema D, Galen JE et al. Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood. J Appl Microbiol 2015; 118:1199–1209 [View Article][PubMed]
    [Google Scholar]
  57. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 2006; 7:3 [View Article][PubMed]
    [Google Scholar]
  58. Thompson KL, Pine PS, Rosenzweig BA, Turpaz Y, Retief J. Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA. BMC Biotechnol 2007; 7:57 [View Article][PubMed]
    [Google Scholar]
  59. Gallego Romero I, Pai AA, Tung J, Gilad Y. RNA-seq: impact of RNA degradation on transcript quantification. BMC Biol 2014; 12:42 [View Article][PubMed]
    [Google Scholar]
  60. Werner AS, Cobbs CG, Kaye D, Hook EW. Studies on the bacteremia of bacterial endocarditis. Jama 1967; 202:199–203 [View Article][PubMed]
    [Google Scholar]
  61. Henry NK, McLimans CA, Wright AJ, Thompson RL, Wilson WR et al. Microbiological and clinical evaluation of the isolator lysis-centrifugation blood culture tube. J Clin Microbiol 1983; 17:864–869[PubMed]
    [Google Scholar]
  62. Dietzman DE, Fischer GW, Schoenknecht FD. Neonatal Escherichia coli septicemia—bacterial counts in blood. J Pediatr 1974; 85:128–130 [View Article][PubMed]
    [Google Scholar]
  63. Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M et al. Quantifying E. coli proteome and transcriptome with Single-Molecule sensitivity in single cells. Science 2010; 329:533–538 [View Article]
    [Google Scholar]
  64. Zhou L, Pollard AJ. A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples. BMC Infect Dis 2012; 12:164 [View Article][PubMed]
    [Google Scholar]
  65. Loonen AJM, Bos MP, van Meerbergen B, Neerken S, Catsburg A et al. Comparison of pathogen DNA isolation methods from large volumes of whole blood to improve molecular diagnosis of bloodstream infections. PLoS One 2013; 8:e72349 [View Article][PubMed]
    [Google Scholar]
  66. Andreasen D, Fog JU, Biggs W, Salomon J, Dahslveen IK et al. Improved microRNA quantification in total RNA from clinical samples. Methods 2010; 50:S6–S9 [View Article][PubMed]
    [Google Scholar]
  67. Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ et al. Evaluation of digital PCR for absolute DNA quantification. Anal Chem 2011; 83:6474–6484 [View Article][PubMed]
    [Google Scholar]
  68. Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics 2005; 21:389–395 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000439
Loading
/content/journal/jmm/10.1099/jmm.0.000439
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error