Fecal Host Transcriptomics for Non-invasive Human Mucosal Immune Profiling: Proof of Concept in Clostridium difficile Infection

Main Article Content

Robert Schlaberg
Amanda Barrett
Kornelia Edes
Michael Graves
Litty Paul
Jenna Rychert
Bert K. Lopansri
Daniel T. Leung


Background: Host factors play an important role in pathogenesis and disease outcome in Clostridium difficile infection (CDI), and characterization of these responses could uncover potential host biomarkers to complement existing microbe-based diagnostics.

Methods: We extracted RNA from fecal samples of patients with CDI and profiled human mRNA using amplicon-based next-generation sequencing (NGS). We compared the fecal host mRNA transcript expression profiles of patients with CDI to controls with non-CDI diarrhea.

Results: We found that the ratio of human actin gamma 1 (ACTG1) to 16S ribosomal RNA (rRNA) was highly correlated with NGS quality as measured by percentage of reads on target. Patients with CDI could be differentiated from those with non-CDI diarrhea based on their fecal mRNA expression profiles using principal component analysis. Among the most differentially expressed genes were ones related to immune response (IL23A, IL34) and actin-cytoskeleton function (TNNT1, MYL4, SMTN, MYBPC3, all adjusted P-values <1×10-3).

Conclusions: In this proof-of-concept study, we used host fecal transcriptomics for non-invasive profiling of the mucosal immune response in CDI. We identified differentially expressed genes with biological plausibility based on animal and cell culture models. This demonstrates the potential of fecal transcriptomics to uncover host-based biomarkers for enteric infections.


Download data is not yet available.

Article Details



1. Lessa FC, Mu Y, Bamberg WM, Beldavs ZG, Dumyati GK, Dunn JR, Farley MM, Holzbauer SM, Meek JI, Phipps EC, Wilson LE, Winston LG, Cohen JA, Limbago BM, Fridkin SK, Gerding DN, McDonald LC. Burden of Clostridium difficile infection in the United States. N Engl J Med. 2015;372(9):825-34. PubMed PMID: 25714160. doi: 10.1056/NEJMoa1408913

2. Chitnis AS, Holzbauer SM, Belflower RM, Winston LG, Bamberg WM, Lyons C, Farley MM, Dumyati GK, Wilson LE, Beldavs ZG, Dunn JR, Gould LH, MacCannell DR, Gerding DN, McDonald LC, Lessa FC. Epidemiology of community-associated Clostridium difficile infection, 2009 through 2011. JAMA Intern Med. 2013;173(14):1359-67. PubMed PMID: 23780507. doi: 10.1001/jamainternmed.2013.7056

3. Khanna S, Pardi DS, Aronson SL, Kammer PP, Orenstein R, St Sauver JL, Harmsen WS, Zinsmeister AR. The epidemiology of community-acquired Clostridium difficile infection: a population-based study. Am J Gastroenterol. 2012;107(1):89-95. PubMed PMID: 22108454. Pubmed Central PMCID: PMC3273904. doi: 10.1038/ajg.2011.398

4. Longtin Y, Trottier S, Brochu G, Paquet-Bolduc B, Garenc C, Loungnarath V, Beaulieu C, Goulet D, Longtin J. Impact of the type of diagnostic assay on Clostridium difficile infection and complication rates in a mandatory reporting program. Clin Infect Dis. 2013;56(1):67-73. PubMed PMID: 23011147. doi: 10.1093/cid/cis840

5. Williamson DA, Basu I, Freeman J, Swager T, Roberts SA. Improved detection of toxigenic Clostridium difficile using the Cepheid Xpert C difficile assay and impact on C difficile infection rates in a tertiary hospital: a double-edged sword. Am J Infect Control. 2013;41(3):270-2. PubMed PMID: 22939418. doi: 10.1016/j.ajic.2012.03.025

6. Usacheva EA, Jin JP, Peterson LR. Host response to Clostridium difficile infection: Diagnostics and detection. J Glob Antimicrob Resist. 2016;7:93-101. PubMed PMID: 27693863. doi: 10.1016/j.jgar.2016.08.002

7. Swale A, Miyajima F, Roberts P, Hall A, Little M, Beadsworth MB, Beeching NJ, Kolamunnage-Dona R, Parry CM, Pirmohamed M. Calprotectin and lactoferrin faecal levels in patients with Clostridium difficile infection (CDI): a prospective cohort study. PLoS One. 2014;9(8):e106118. PubMed PMID: 25170963. Pubmed Central PMCID: 4149523. doi: 10.1371/journal.pone.0106118

8. Loo VG, Bourgault AM, Poirier L, Lamothe F, Michaud S, Turgeon N, Toye B, Beaudoin A, Frost EH, Gilca R, Brassard P, Dendukuri N, Beliveau C, Oughton M, Brukner I, Dascal A. Host and pathogen factors for Clostridium difficile infection and colonization. N Engl J Med. 2011;365(18):1693-703. PubMed PMID: 22047560. doi: 10.1056/NEJMoa1012413

9. Hasegawa M, Yamazaki T, Kamada N, Tawaratsumida K, Kim YG, Nunez G, Inohara N. Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen. J Immunol. 2011;186(8):4872-80. PubMed PMID: 21411735. doi: 10.4049/jimmunol.1003761

10. Jarchum I, Liu M, Shi C, Equinda M, Pamer EG. Critical role for MyD88-mediated neutrophil recruitment during Clostridium difficile colitis. Infect Immun. 2012;80(9):2989-96. PubMed PMID: 22689818. Pubmed Central PMCID: PMC3418725. doi: 10.1128/IAI.00448-12

11. Ryan A, Lynch M, Smith SM, Amu S, Nel HJ, McCoy CE, Dowling JK, Draper E, O'Reilly V, McCarthy C, O'Brien J, Ni Eidhin D, O'Connell MJ, Keogh B, Morton CO, Rogers TR, Fallon PG, O'Neill LA, Kelleher D, Loscher CE. A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins. PLoS Pathog. 2011;7(6):e1002076. PubMed PMID: 21738466. Pubmed Central PMCID: PMC3128122. doi: 10.1371/journal.ppat.1002076

12. Abt MC, Lewis BB, Caballero S, Xiong H, Carter RA, Susac B, Ling L, Leiner I, Pamer EG. Innate Immune Defenses Mediated by Two ILC Subsets Are Critical for Protection against Acute Clostridium difficile Infection. Cell Host Microbe. 2015;18(1):27-37. PubMed PMID: 26159718. Pubmed Central PMCID: PMC4537644. doi: 10.1016/j.chom.2015.06.011

13. Buonomo EL, Madan R, Pramoonjago P, Li L, Okusa MD, Petri WA, Jr. Role of interleukin 23 signaling in Clostridium difficile colitis. J Infect Dis. 2013;208(6):917-20. PubMed PMID: 23776194. Pubmed Central PMCID: PMC3749013. doi: 10.1093/infdis/jit277

14. Kyne L, Warny M, Qamar A, Kelly CP. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N Engl J Med. 2000;342(6):390-7. PubMed PMID: 10666429. doi: 10.1056/NEJM200002103420604

15. Kyne L, Warny M, Qamar A, Kelly CP. Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. Lancet. 2001;357(9251):189-93. PubMed PMID: 11213096. doi: 10.1016/S0140-6736(00)03592-3

16. Garey KW, Jiang ZD, Ghantoji S, Tam VH, Arora V, Dupont HL. A common polymorphism in the interleukin-8 gene promoter is associated with an increased risk for recurrent Clostridium difficile infection. Clin Infect Dis. 2010;51(12):1406-10. PubMed PMID: 21058913. doi: 10.1086/657398

17. El Feghaly RE, Stauber JL, Deych E, Gonzalez C, Tarr PI, Haslam DB. Markers of intestinal inflammation, not bacterial burden, correlate with clinical outcomes in Clostridium difficile infection. Clin Infect Dis. 2013;56(12):1713-21. PubMed PMID: 23487367. Pubmed Central PMCID: 3707425. 10.1093/cid/cit147

18. El Feghaly RE, Stauber JL, Tarr PI, Haslam DB. Intestinal inflammatory biomarkers and outcome in pediatric Clostridium difficile infections. J Pediatr. 2013;163(6):1697-704 e2. PubMed PMID: 24011765. Pubmed Central PMCID: 4098967. doi: 10.1016/j.jpeds.2013.07.029

19. Cernada M, Bauerl C, Serna E, Collado MC, Martinez GP, Vento M. Sepsis in preterm infants causes alterations in mucosal gene expression and microbiota profiles compared to non-septic twins. Sci Rep. 2016;6:25497. PubMed PMID: 27180802. Pubmed Central PMCID: 4867619. doi: 10.1038/srep25497

20. Knight JM, Davidson LA, Herman D, Martin CR, Goldsby JS, Ivanov IV, Donovan SM, Chapkin RS. Non-invasive analysis of intestinal development in preterm and term infants using RNA-Sequencing. Sci Rep. 2014;4:5453. PubMed PMID: 24965658. Pubmed Central PMCID: 4071321. doi: 10.1038/srep05453

21. Stauber J, Shaikh N, Ordiz MI, Tarr PI, Manary MJ. Droplet digital PCR quantifies host inflammatory transcripts in feces reliably and reproducibly. Cell Immunol. 2016;303:43-9. PubMed PMID: 27063479. Pubmed Central PMCID: 4863679. doi: 10.1016/j.cellimm.2016.03.007

22. Fujitani S, George WL, Murthy AR. Comparison of clinical severity score indices for Clostridium difficile infection. Infect Control Hosp Epidemiol. 2011;32(3):220-8. PubMed PMID: 21460506. doi: 10.1086/658336

23. Zar FA, Bakkanagari SR, Moorthi KM, Davis MB. A comparison of vancomycin and metronidazole for the treatment of Clostridium difficile-associated diarrhea, stratified by disease severity. Clin Infect Dis. 2007;45(3):302-7. PubMed PMID: 17599306. doi: 10.1086/519265

24. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. PubMed PMID: 25516281. Pubmed Central PMCID: PMC4302049. doi: 10.1186/s13059-014-0550-8

25. Flygare S, Simmon K, Miller C, Qiao Y, Kennedy B, Di Sera T, Graf EH, Tardif KD, Kapusta A, Rynearson S, Stockmann C, Queen K, Tong S, Voelkerding KV, Blaschke A, Byington CL, Jain S, Pavia A, Ampofo K, Eilbeck K, Marth G, Yandell M, Schlaberg R. Taxonomer: an interactive metagenomics analysis portal for universal pathogen detection and host mRNA expression profiling. Genome Biol. 2016;17(1):111. PubMed PMID: 27224977. Pubmed Central PMCID: PMC4880956. doi: 10.1186/s13059-016-0969-1

26. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44-57. PubMed PMID: 19131956. doi: 10.1038/nprot.2008.211

27. Stockmann C, Rogatcheva M, Harrel B, Vaughn M, Crisp R, Poritz M, Thatcher S, Korgenski EK, Barney T, Daly J, Pavia AT. How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection. Clin Microbiol Infect. 2015;21(2):179 e9-15. PubMed PMID: 25599941. Pubmed Central PMCID: PMC4330102. doi: 10.1016/j.cmi.2014.07.011

28. Truong CY, Gombar S, Wilson R, Sundararajan G, Tekic N, Holubar M, Shepard J, Madison A, Tompkins L, Shah N, Deresinski S, Schroeder LF, Banaei N. Real-Time Electronic Tracking of Diarrheal Episodes and Laxative Therapy Enables Verification of Clostridium difficile Clinical Testing Criteria and Reduction of Clostridium difficile Infection Rates. J Clin Microbiol. 2017;55(5):1276-84. PubMed PMID: 28250001. Pubmed Central PMCID: PMC5405247. doi: 10.1128/JCM.02319-16

29. Cowardin CA, Kuehne SA, Buonomo EL, Marie CS, Minton NP, Petri WA, Jr. Inflammasome activation contributes to interleukin-23 production in response to Clostridium difficile. MBio. 2015;6(1). PubMed PMID: 25626905. Pubmed Central PMCID: PMC4324312. doi: 10.1128/mBio.02386-14

30. McDermott AJ, Falkowski NR, McDonald RA, Pandit CR, Young VB, Huffnagle GB. Interleukin-23 (IL-23), independent of IL-17 and IL-22, drives neutrophil recruitment and innate inflammation during Clostridium difficile colitis in mice. Immunology. 2016;147(1):114-24. PubMed PMID: 26455347. Pubmed Central PMCID: PMC4693884. doi: 10.1111/imm.12545

31. Zwicker S, Martinez GL, Bosma M, Gerling M, Clark R, Majster M, Soderman J, Almer S, Bostrom EA. Interleukin 34: a new modulator of human and experimental inflammatory bowel disease. Clin Sci (Lond). 2015;129(3):281-90. PubMed PMID: 25896238. Pubmed Central PMCID: PMC4557398. doi: 10.1042/CS20150176

32. Franze E, Monteleone I, Cupi ML, Mancia P, Caprioli F, Marafini I, Colantoni A, Ortenzi A, Laudisi F, Sica G, Sileri P, Pallone F, Monteleone G. Interleukin-34 sustains inflammatory pathways in the gut. Clin Sci (Lond). 2015;129(3):271-80. PubMed PMID: 25800277. doi: 10.1042/CS20150132

33. Franze E, Marafini I, De Simone V, Monteleone I, Caprioli F, Colantoni A, Ortenzi A, Crescenzi F, Izzo R, Sica G, Sileri P, Rossi P, Pallone F, Monteleone G. Interleukin-34 Induces Cc-chemokine Ligand 20 in Gut Epithelial Cells. J Crohns Colitis. 2016;10(1):87-94. PubMed PMID: 26449789. 10.1093/ecco-jcc/jjv181

34. Just I, Selzer J, Wilm M, von Eichel-Streiber C, Mann M, Aktories K. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature. 1995;375(6531):500-3. PubMed PMID: 7777059. doi: 10.1038/375500a0

35. Riegler M, Sedivy R, Pothoulakis C, Hamilton G, Zacherl J, Bischof G, Cosentini E, Feil W, Schiessel R, LaMont JT, et al. Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro. J Clin Invest. 1995;95(5):2004-11. PubMed PMID: 7738167. Pubmed Central PMCID: PMC295778. doi: 10.1172/JCI117885

36. Popoff MR, Rubin EJ, Gill DM, Boquet P. Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain. Infect Immun. 1988;56(9):2299-306. PubMed PMID: 3137166. Pubmed Central PMCID: PMC259564.

37. Swindells J, Brenwald N, Reading N, Oppenheim B. Evaluation of diagnostic tests for Clostridium difficile infection. J Clin Microbiol. 2010;48(2):606-8. PubMed PMID: 20032256. Pubmed Central PMCID: PMC2815642. doi: 10.1128/JCM.01579-09

38. Planche TD, Davies KA, Coen PG, Finney JM, Monahan IM, Morris KA, O'Connor L, Oakley SJ, Pope CF, Wren MW, Shetty NP, Crook DW, Wilcox MH. Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection. Lancet Infect Dis. 2013;13(11):936-45. PubMed PMID: 24007915. Pubmed Central PMCID: 3822406. doi: 10.1016/S1473-3099(13)70200-7

39. Dubberke ER, Han Z, Bobo L, Hink T, Lawrence B, Copper S, Hoppe-Bauer J, Burnham CA, Dunne WM, Jr. Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections. J Clin Microbiol. 2011;49(8):2887-93. PubMed PMID: 21697328. Pubmed Central PMCID: PMC3147743. doi: 10.1128/JCM.00891-11

40. Polage CR, Gyorke CE, Kennedy MA, Leslie JL, Chin DL, Wang S, Nguyen HH, Huang B, Tang YW, Lee LW, Kim K, Taylor S, Romano PS, Panacek EA, Goodell PB, Solnick JV, Cohen SH. Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era. JAMA Intern Med. 2015;175(11):1792-801. PubMed PMID: 26348734. Pubmed Central PMCID: 4948649. doi: 10.1001/jamainternmed.2015.4114

41. McDonald LC, Gerding DN, Johnson S, Bakken JS, Carroll KC, Coffin SE, Dubberke ER, Garey KW, Gould CV, Kelly C, Loo V, Shaklee Sammons J, Sandora TJ, Wilcox MH. Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis. 2018;66(7):e1-e48. PubMed PMID: 29462280. doi: 10.1093/cid/cix1085

42. Ahmed FE, James SI, Lysle DT, Dobbs LJ, Jr., Johnke RM, Flake G, Stockton P, Sinar DR, Naziri W, Evans MJ, Kovacs CJ, Allison RR. Improved methods for extracting RNA from exfoliated human colonocytes in stool and RT-PCR analysis. Dig Dis Sci. 2004;49(11-12):1889-98. PubMed PMID: 15628722.

43. Alexander RJ, Raicht RF. Purification of total RNA from human stool samples. Dig Dis Sci. 1998;43(12):2652-8. PubMed PMID: 9881497.

44. Bennett WE, Jr., Gonzalez-Rivera R, Shaikh N, Magrini V, Boykin M, Warner BB, Hamvas A, Tarr PI. A method for isolating and analyzing human mRNA from newborn stool. J Immunol Methods. 2009;349(1-2):56-60. PubMed PMID: 19660464. Pubmed Central PMCID: PMC2850193. doi: 10.1016/j.jim.2009.07.013

45. Reck M, Tomasch J, Deng Z, Jarek M, Husemann P, Wagner-Dobler I, Consortium C. Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation. BMC Genomics. 2015;16:494. PubMed PMID: 26140923. Pubmed Central PMCID: 4490624. doi: 10.1186/s12864-015-1694-y

46. Giannoukos G, Ciulla DM, Huang K, Haas BJ, Izard J, Levin JZ, Livny J, Earl AM, Gevers D, Ward DV, Nusbaum C, Birren BW, Gnirke A. Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes. Genome Biol. 2012;13(3):R23. PubMed PMID: 22455878. Pubmed Central PMCID: PMC3439974. doi: 10.1186/gb-2012-13-3-r23

47. Li W, Turner A, Aggarwal P, Matter A, Storvick E, Arnett DK, Broeckel U. Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis. BMC Genomics. 2015;16:1069. PubMed PMID: 26673413. Pubmed Central PMCID: PMC4681149. doi: 10.1186/s12864-015-2270-1

48. FitzGerald LM, Jung CH, Wong EM, Joo JE, Gould JA, Vasic V, Bassett JK, O'Callaghan N, Nottle T, Pedersen J, Giles GG, Southey MC. Obtaining high quality transcriptome data from formalin-fixed, paraffin-embedded diagnostic prostate tumor specimens. Lab Invest. 2018. PubMed PMID: 29339835. doi: 10.1038/s41374-017-0001-8

49. Bhattacharya S, Rosenberg AF, Peterson DR, Grzesik K, Baran AM, Ashton JM, Gill SR, Corbett AM, Holden-Wiltse J, Topham DJ, Walsh EE, Mariani TJ, Falsey AR. Transcriptomic Biomarkers to Discriminate Bacterial from Nonbacterial Infection in Adults Hospitalized with Respiratory Illness. Sci Rep. 2017;7(1):6548. PubMed PMID: 28747714. Pubmed Central PMCID: PMC5529430. doi: 10.1038/s41598-017-06738-3

50. McClain MT, Nicholson BP, Park LP, Liu TY, Hero AO, 3rd, Tsalik EL, Zaas AK, Veldman T, Hudson LL, Lambkin-Williams R, Gilbert A, Burke T, Nichols M, Ginsburg GS, Woods CW. A Genomic Signature of Influenza Infection Shows Potential for Presymptomatic Detection, Guiding Early Therapy, and Monitoring Clinical Responses. Open Forum Infect Dis. 2016;3(1):ofw007. PubMed PMID: 26933666. Pubmed Central PMCID: PMC4771939. doi: 10.1093/ofid/ofw007

51. Ramilo O, Mejias A, Mahajan P, Kuppermann N. RNA signature test to distinguish bacterial from viral infection. J Pediatr. 2017;182:401-4. PubMed PMID: 28237452. doi: 10.1016/j.jpeds.2016.12.066

52. Tsalik EL, Henao R, Nichols M, Burke T, Ko ER, McClain MT, Hudson LL, Mazur A, Freeman DH, Veldman T, Langley RJ, Quackenbush EB, Glickman SW, Cairns CB, Jaehne AK, Rivers EP, Otero RM, Zaas AK, Kingsmore SF, Lucas J, Fowler VG, Jr., Carin L, Ginsburg GS, Woods CW. Host gene expression classifiers diagnose acute respiratory illness etiology. Sci Transl Med. 2016;8(322):322ra11. PubMed PMID: 26791949. Pubmed Central PMCID: PMC4905578. doi: 10.1126/scitranslmed.aad6873

53. Zaas AK, Burke T, Chen M, McClain M, Nicholson B, Veldman T, Tsalik EL, Fowler V, Rivers EP, Otero R, Kingsmore SF, Voora D, Lucas J, Hero AO, Carin L, Woods CW, Ginsburg GS. A host-based RT-PCR gene expression signature to identify acute respiratory viral infection. Sci Transl Med. 2013;5(203):203ra126. PubMed PMID: 24048524. Pubmed Central PMCID: PMC4286889. doi: 10.1126/scitranslmed.3006280

Most read articles by the same author(s)