Medical Microbiology and Immunology

, Volume 204, Issue 6, pp 647–656 | Cite as

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment

  • Nair Nazareth
  • Fernando Magro
  • Rui Appelberg
  • Jani Silva
  • Daniela Gracio
  • Rosa Coelho
  • José Miguel Cabral
  • Candida Abreu
  • Guilherme Macedo
  • Tim J. Bull
  • Amélia Sarmento
Original Investigation


Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn’s disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.


Inflammatory bowel disease Mycobacterium avium subsp. paratuberculosis Macrophages Phagosomal maturation 



This work was funded by the Portuguese Science Foundation (FCT) and COMPETE Program subsidized by FEDER, through the PIC/IC/82802/2007 Grant. The monoclonal antibody H4A3-c (antihuman LAMP-1), developed by J. Thomas August and James E. K. Hildreth, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242. The authors wish to thank Paula Sampaio, for helping with confocal image acquisition and processing. The authors also wish to thank all patients and controls for collaborating in this study.

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Pickup RW, Rhodes G, Arnott S, Sidi-Boumedine K, Bull TJ, Weightman A, Hurley M, Hermon-Taylor J (2005) Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn’s disease cases in the city of Cardiff. Appl Environ Microbiol 71(4):2130–2139PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Chacon O, Bermudez LE, Barletta RG (2004) Johne’s disease, inflammatory bowel disease, and Mycobacterium paratuberculosis. Ann Rev Microbiol 58:329–363CrossRefGoogle Scholar
  3. 3.
    Over K, Crandall PG, O’Bryan CA, Ricke SC (2011) Current perspectives on Mycobacterium avium subsp. paratuberculosis, Johne’s disease, and Crohn’s disease: a review. Crit Rev Microbiol 37(2):141–156CrossRefPubMedGoogle Scholar
  4. 4.
    Kasperbauer SH, Daley CL (2008) Diagnosis and treatment of infections due to Mycobacterium avium complex. Sem Respir Crit Care Med 29(5):569–576CrossRefGoogle Scholar
  5. 5.
    Agdestein A, Jones A, Flatberg A, Johansen TB, Heffernan IA, Djønne B, Bosco A, Olsen I (2014) Intracellular growth of Mycobacterium avium subspecies and global transcriptional responses in human macrophages after infection. BMC Genom 15(1):1–14CrossRefGoogle Scholar
  6. 6.
    Rindi L, Garzelli C (2014) Genetic diversity and phylogeny of Mycobacterium avium. Infect Gen Evol 21(1):375–383CrossRefGoogle Scholar
  7. 7.
    Glawischnig W, Steineck T, Spergser J (2006) Infections caused by Mycobacterium avium subspecies avium, hominissuis, and paratuberculosis in free-ranging red deer (Cervus elaphus hippelaphus) in Austria, 2001-2004. J Wild Dis 42(4):724–731CrossRefGoogle Scholar
  8. 8.
    Judge J, Kyriazakis I, Greig A, Allcroft DJ, Hutchings MR (2005) Clustering of Mycobacterium avium subsp. paratuberculosis in rabbits and the environment: how hot is a hot spot? Appl Environ Microbiol 71(10):6033–6038PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    McClure HM, Chiodini RJ, Anderson DC, Swenson RB, Thayer WR, Coutu JA (1987) Mycobacterium paratuberculosis infection in a colony of stumptail macaques (Macaca arctoides). J Infect Dis 155(5):1011–1019CrossRefPubMedGoogle Scholar
  10. 10.
    Abubakar I, Myhill D, Aliyu SH, Hunter PR (2008) Detection of Mycobacterium avium subspecies paratuberculosis from patients with Crohn’s disease using nucleic acid-based techniques: a systematic review and meta-analysis. Inflamm Bowel Dis 14(3):401–410CrossRefPubMedGoogle Scholar
  11. 11.
    Bull TJ, McMinn EJ, Sidi-Boumedine K et al (2003) Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn’s disease. J Clin Microbiol 41(7):2915–2923PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Mendoza JL, San-Pedro A, Culebras E et al (2010) High prevalence of viable Mycobacterium avium subspecies paratuberculosis in Crohn’s disease. World J Gastroenterol 16(36):4558–4563PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Secott TE, Lin TL, Wu CC (2004) Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins. Infect Immun 72(7):3724–3732PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Golan L, Livneh-Kol A, Gonen E, Yagel S, Rosenshine I, Shpigel NY (2009) Mycobacterium avium paratuberculosis invades human small-intestinal goblet cells and elicits inflammation. J Infect Dis 199(3):350–354CrossRefPubMedGoogle Scholar
  15. 15.
    Weiss DJ, Souza CD (2008) Review paper: modulation of mononuclear phagocyte function by Mycobacterium avium subsp. paratuberculosis. Vet Pathol 45(6):829–841CrossRefPubMedGoogle Scholar
  16. 16.
    Hume DA (2008) Macrophages as APC and the dendritic cell myth. J Immunol 181(9):5829–5835CrossRefPubMedGoogle Scholar
  17. 17.
    Sheikh SZ, Plevy SE (2010) The role of the macrophage in sentinel responses in intestinal immunity. Curr Opin Gastroenterol 26(6):578–582PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Cosma CL, Sherman DR, Ramakrishnan L (2003) The secret lives of the pathogenic mycobacteria. Ann Rev Microbiol 57:641–676CrossRefGoogle Scholar
  19. 19.
    Pieters J, Gatfield J (2002) Hijacking the host: survival of pathogenic mycobacteria inside macrophages. Trends Microbiol 10(3):142–146CrossRefPubMedGoogle Scholar
  20. 20.
    Abendaño N, Juste RA, Alonso-Hearn M (2013) Anti-inflammatory and antiapoptotic responses to infection: a common denominator of human and bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis. BioMed Res Intern 2:1–7CrossRefGoogle Scholar
  21. 21.
    Kabara E, Coussens PM (2012) Infection of primary bovine macrophages with Mycobacterium avium subspecies paratuberculosis suppresses host cell apoptosis. Front Microbiol 3:1–10CrossRefGoogle Scholar
  22. 22.
    Rumsey J, Valentine JF, Naser SA (2006) Inhibition of phagosome maturation and survival of Mycobacterium avium subspecies paratuberculosis in polymorphonuclear leukocytes from Crohn’s disease patients. Med Sci Monit 12(4):BR130–BR139PubMedGoogle Scholar
  23. 23.
    Keown DA, Collings DA, Keenan JI (2012) Uptake and persistence of Mycobacterium avium subsp. paratuberculosis in human monocytes. Infect Immun 80(11):3768–3775PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Hostetter J, Steadham E, Haynes J, Bailey T, Cheville N (2003) Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells. Comp Immunol Microbiol Infect Dis 26(4):269–283CrossRefPubMedGoogle Scholar
  25. 25.
    Weiss DJ, Evanson OA, Deng M, Abrahamsen MS (2004) Gene expression and antimicrobial activity of bovine macrophages in response to Mycobacterium avium subsp. paratuberculosis. Vet Pathol 41(4):326–337CrossRefPubMedGoogle Scholar
  26. 26.
    Kuehnel MP, Goethe R, Habermann A, Mueller E, Rohde M, Griffiths G, Valentin-Weigand P (2001) Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. Cell Microbiol 3(8):551–566CrossRefPubMedGoogle Scholar
  27. 27.
    Bull TJ, Linedale R, Hinds J, Hermon-Taylor J (2009) A rhodanine agent active against non-replicating intracellular Mycobacterium avium subspecies paratuberculosis. Gut Pathog 1:25–36PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Smith AM, Rahman FZ, Hayee BH et al (2009) Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn’s disease. J Exp Med 206(9):1883–1897PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Sewell GW, Rahman FZ, Levine AP et al (2012) Defective tumor necrosis factor release from Crohnʼs disease macrophages in response to toll-like receptor activation: relationship to phenotype and genome-wide association susceptibility loci. Inflamm Bowel Dis 18(11):2120–2127PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Magro F, Portela F (2010) Management of inflammatory bowel disease with infliximab and other anti-tumor necrosis factor alpha therapies. BioDrugs 24(Suppl 1):3–14CrossRefPubMedGoogle Scholar
  31. 31.
    Dignass A, Van Assche G, Lindsay JO et al (2010) The second European evidence-based consensus on the diagnosis and management of Crohn’s disease: current management. J Crohns Colitis 4(1):28–62CrossRefPubMedGoogle Scholar
  32. 32.
    Dignass A, Eliakim R, Magro F et al (2012) Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: definitions and diagnosis. J Crohns Colitis 6:965–990CrossRefPubMedGoogle Scholar
  33. 33.
    Scanu AM, Bull TJ, Cannas S, Sanderson JD, Sechi LA, Dettori G, Zanetti S, Hermon-Taylor J (2007) Mycobacterium avium subspecies paratuberculosis infection in cases of irritable bowel syndrome and comparison with Crohn’s disease and Johne’s disease: common neural and immune pathogenicities. J Clin Microbiol 45(12):3883–3890PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Hermon-Taylor J (2001) Protagonist: Mycobacterium avium subspecies paratuberculosis is a cause of Crohn’s disease. Gut 49(6):755–756PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Sechi LA, Scanu AM, Molicotti P, Cannas S, Mura M, Dettori G, Fadda G, Zanetti S (2005) Detection and Isolation of Mycobacterium avium subspecies paratuberculosis from intestinal mucosal biopsies of patients with and without Crohn’s disease in Sardinia. Am J Gastroenterol 100(7):1529–1536CrossRefPubMedGoogle Scholar
  36. 36.
    Patel A, Shah N (2011) Mycobacterium avium subsp. paratuberculosis incidences in milk and milk products, their isolation, enumeration, characterization, and role in human health. J Microbiol Immunol Infect 44(6):473–479CrossRefPubMedGoogle Scholar
  37. 37.
    Gumber S, Taylor DL, Marsh IB, Whittington RJ (2009) Growth pattern and partial proteome of Mycobacterium avium subsp. paratuberculosis during the stress response to hypoxia and nutrient starvation. Vet Microbiol 133(4):344–357CrossRefPubMedGoogle Scholar
  38. 38.
    Gumber S, Whittington RJ (2009) Analysis of the growth pattern, survival and proteome of Mycobacterium avium subsp. paratuberculosis following exposure to heat. Vet Microbiol 136(1–2):82–90CrossRefPubMedGoogle Scholar
  39. 39.
    Cosnes J (2004) Tobacco and IBD: relevance in the understanding of disease mechanisms and clinical practice. Best Pract Res Clin Gastroenterol 18(3):481–496CrossRefPubMedGoogle Scholar
  40. 40.
    Shleeva MO, Salina EG, Kaprelyants AS (2010) Dormant forms of mycobacteria. Microbiology 79(1):1–12CrossRefGoogle Scholar
  41. 41.
    Manabe YC, Bishai WR (2000) Latent Mycobacterium tuberculosis-persistence, patience, and winning by waiting. Nat Med 6(12):1327–1329CrossRefPubMedGoogle Scholar
  42. 42.
    Gengenbacher M, Kaufmann SHE (2012) Mycobacterium tuberculosis: success through dormancy. FEMS Microbiol Rev 36(3):514–532PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    Lamont EA, Bannantine JP, Armién A, Ariyakumar DS, Sreevatsan S (2012) Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures. PLoS ONE 7(1):e30648PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    Bruns H, Meinken C, Schauenberg P, Härter G, Kern P, Modlin RL, Antoni C, Stenger S (2009) Anti-TNF immunotherapy reduces CD8+ T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans. J Clin Invest 119(5):1167–1177PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Boschetti G, Nancey S, Sardi F, Roblin X, Flourié B, Kaiserlian D (2011) Therapy with anti-TNFα antibody enhances number and function of Foxp3+ regulatory T cells in inflammatory bowel diseases. Inflamm Bowel Dis 17(1):160–170CrossRefPubMedGoogle Scholar
  46. 46.
    Seong S-S, Choi C-B, Woo J-H et al (2007) Incidence of tuberculosis in Korean patients with rheumatoid arthritis (RA): effects of RA itself and of tumor necrosis factor blockers. J Rheumatol 34(4):706–711PubMedGoogle Scholar
  47. 47.
    Naser SA, Ghobrial G, Romero C, Valentine JF (2004) Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet 364(9439):1039–1044CrossRefPubMedGoogle Scholar
  48. 48.
    Nazareth N, Magro F, Silva J et al (2014) Infliximab therapy increases the frequency of circulating CD16+ monocytes and modifies macrophage cytokine response to bacterial infection. Clin Exp Immunol 177(3):703–711PubMedCentralCrossRefPubMedGoogle Scholar
  49. 49.
    Pierce ES (2010) Ulcerative colitis and Crohn’s disease: is Mycobacterium avium subspecies paratuberculosis the common villain? Gut Pathog 2(1):21–32PubMedCentralCrossRefPubMedGoogle Scholar
  50. 50.
    Homer CR, Richmond AL, Rebert NA, Achkar JP, Mcdonald C (2010) ATG16L1 and NOD2 Interact in an Autophagy-Dependent Antibacterial Pathway Implicated in Crohn’s Disease Pathogenesis. Gastroenterology 139(5):1630–1641PubMedCentralCrossRefPubMedGoogle Scholar
  51. 51.
    Lapaquette P, Brest P, Hofman P, Darfeuille-Michaud A (2012) Etiology of Crohn’s disease: many roads lead to autophagy. J Mol Med 90(9):987–996CrossRefPubMedGoogle Scholar
  52. 52.
    Koo IC, Wang C, Raghavan S, Morisaki JH, Cox JS, Brown EJ (2008) ESX-1-dependent cytolysis in lysosome secretion and inflammasome activation during mycobacterial infection. Cell Microbiol 10(9):1866–1878PubMedCentralCrossRefPubMedGoogle Scholar
  53. 53.
    Lamont EA, O’Grady SM, Davis WC, Eckstein T, Sreevatsan S (2012) Infection with Mycobacterium avium subsp. paratuberculosis results in rapid interleukin-1 release and macrophage transepithelial migration. Infect Immun 80(9):3225–3235PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Nair Nazareth
    • 1
  • Fernando Magro
    • 2
    • 3
    • 4
  • Rui Appelberg
    • 5
    • 6
  • Jani Silva
    • 1
  • Daniela Gracio
    • 2
    • 4
  • Rosa Coelho
    • 3
  • José Miguel Cabral
    • 2
    • 4
  • Candida Abreu
    • 6
    • 7
  • Guilherme Macedo
    • 3
  • Tim J. Bull
    • 8
  • Amélia Sarmento
    • 1
    • 5
  1. 1.FP-ENAS (UFP Energy, Environment and Health Research Unit), CEBIMED (Biomedical Research Centre)University Fernando PessoaPortoPortugal
  2. 2.Institute of Pharmacology and Therapeutics, Faculdade de MedicinaUniversidade do PortoPortoPortugal
  3. 3.Gastroenterology DepartmentCentro Hospitalar S. JoãoPortoPortugal
  4. 4.MedInUP - Center for Drug Discovery and Innovative MedicinesUniversidade do PortoPortoPortugal
  5. 5.Infection and Immunity Unit, IBMC – Instituto de Biologia Molecular e CelularUniversidade do PortoPortoPortugal
  6. 6.Department of Infectious DiseasesCentro Hospitalar S. JoãoPortoPortugal
  7. 7.Nephrology Research and Development Unit, Faculdade de MedicinaUniversidade do PortoPortoPortugal
  8. 8.Infection and Immunity Research InstituteSt George’s University of LondonLondonUK

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