Glycoconjugate Journal

, Volume 36, Issue 5, pp 429–438 | Cite as

Detection and characterization of bacterial polysaccharides in drug-resistant enterococci

  • Liaqat AliEmail author
  • Hubert E. Blum
  • Türkân Sakιnç
Original Article


Enterococcus faecium (E. faecium) has emerged as one of today’s leading causes of health care-associated infections that is difficult to treat with the available antibiotics. These pathogens produce capsular polysaccharides on the cell surface which play a significant role in adhesion, virulence and evasion. Therefore, we aimed at the identification and characterization of bacterial polysaccharide antigens which are central for the development of vaccine-based prophylactic approaches. The crude cell wall-associated polysaccharides from E. faecium, its mutant and complemented strains were purified and analyzed by a primary antibody raised against lipoteichoic acid (LTA) and diheteroglycan (DHG). The resistant E. faecium strains presumably possess novel capsular polysaccharides that allow them to avoid the evasion from opsonic killing. The E. faecium U0317 strain was very well opsonized by anti-U0317 (~95%), an antibody against the whole bacterial cell. The deletion mutant showed a significantly increased susceptibility to opsonophagocytic killing (90–95%) against the penicillin binding protein (anti-PBP-5). By comparison, in a mouse urinary tract and rat endocarditis infection model, respectively, there were no significant differences in virulence. In this study we explored the biological role of the capsule of E. faecium. Our findings showed that the U0317 strain is not only sensitive to anti-LTA but also to antibodies against other enterococcal surface proteins. Our findings demonstrate that polysaccharides capsule mediated-resistance to opsonophagocytosis. We also found that the capsular polysaccharides do not play an important role in bacterial virulence in urinary tract and infective endocarditis in vivo models.


E. faecium Capsular polysaccharide Opsonophagocytosis Virulence 



Capsular polysaccharides


Colony-forming unit



E. faecium

Enterococcus faecium


Lipoteichoic Acid


Opsonophagocytic Assays


Penicillin Binding Protein-5


Urinary Tract Infection


Vancomycin-Resistant Enterococci





LA sincerely thanks the Deutscher Akademischer AustauschDienst (DAAD) for the award of a PhD fellowship and Dominique Wobser for help with the animal experiments.

Authors’ contributions

LA and TS conceived and designed the study. LA analyzed the data and drafted the manuscript. HEB critically reviewed the manuscript. All the authors studied and approved the final manuscript.


This work was supported by grants from the German Ministry of Science and Education (BMBF: UroGenOmics0315833C).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All animal experiments were performed in compliance with the German animal protection law (TierSchG). The mice were housed and handled following good animal practice as defined by FELASA and the national animal welfare body GV-SOLAS. The animal welfare committees of the University of Freiburg (Regierungspräsidium Freiburg Az 35/9185.81/G-11/118 and Az 35/9185.81/G-12/070) approved all animal experiments.

Consent for publication

Not applicable.


  1. 1.
    Costerton, J.W., Irvin, R.T., Cheng, K.J.: The bacterial glycocalyx in nature and disease. Annu. Rev. Microbiol. 35, 299–324 (1981)CrossRefGoogle Scholar
  2. 2.
    Micoli, F., Costantino, P., Adamo, R.: Potential targets for next generation antimicrobial glycoconjugate vaccines. FEMS Microbiol. Rev. 42, 388–423 (2018)CrossRefGoogle Scholar
  3. 3.
    Whitfield, C., Valvano, M.A.: Biosynthesis and expression of cell-surface polysaccharides in gram-negative bacteria. Adv. Microb. Physiol. 35, 135–246 (1993)CrossRefGoogle Scholar
  4. 4.
    Boulnois, G.J., Roberts, I.S.: Genetics of capsular polysaccharide production in bacteria. Curr. Top. Microbiol. Immunol. 150, 1–18 (1990)Google Scholar
  5. 5.
    O’Riordan, K., Lee, J.C.: Staphylococcus aureus capsular polysaccharides. Clin. Microbiol. Rev. 17, 218–234 (2004)CrossRefGoogle Scholar
  6. 6.
    Kadioglu, A., Weiser, J.N., Paton, J.C., Andrew, P.W.: The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat. Rev. Microbiol. 6, 288–301 (2008)CrossRefGoogle Scholar
  7. 7.
    Enterococci: From commensals to leading causes of drug resistant infection - PubMed - NCBI, (2014)Google Scholar
  8. 8.
    Thurlow, L.R., Thomas, V.C., Hancock, L.E.: Capsular polysaccharide production in Enterococcus faecalis and contribution of CpsF to capsule serospecificity. J. Bacteriol. 191, 6203–6210 (2009)CrossRefGoogle Scholar
  9. 9.
    McBride, S.M., Fischetti, V.A., Leblanc, D.J., Moellering, R.C., Gilmore, M.S.: Genetic diversity among Enterococcus faecalis. PLoS One. 2, e582 (2007)CrossRefGoogle Scholar
  10. 10.
    Thurlow, L.R., Thomas, V.C., Fleming, S.D., Hancock, L.E.: Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion. Infect. Immun. 77, 5551–5557 (2009)CrossRefGoogle Scholar
  11. 11.
    Ali, L., Spiess, M., Wobser, D., Rodriguez, M., Blum, H.E., Sakιnç, T.: Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317. Infect. Genet. Evol. 37, 215–224 (2016)CrossRefGoogle Scholar
  12. 12.
    Bentley, S.D., Aanensen, D.M., Mavroidi, A., Saunders, D., Rabbinowitsch, E., Collins, M., Donohoe, K., Harris, D., Murphy, L., Quail, M.A., Samuel, G., Skovsted, I.C., Kaltoft, M.S., Barrell, B., Reeves, P.R., Parkhill, J., Spratt, B.G.: Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PLoS Genet. 2, e31 (2006)CrossRefGoogle Scholar
  13. 13.
    Pan, Y.-J., Lin, T.-L., Chen, C.-T., Chen, Y.-Y., Hsieh, P.-F., Hsu, C.-R., Wu, M.-C., Wang, J.-T.: Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp. Sci. Rep. 5, 15573 (2015)CrossRefGoogle Scholar
  14. 14.
    Palmer, K.L., Godfrey, P., Griggs, A., Kos, V.N., Zucker, J., Desjardins, C., Cerqueira, G., Gevers, D., Walker, S., Wortman, J., Feldgarden, M., Haas, B., Birren, B., Gilmore, M.S.: Comparative genomics of enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defining characteristics of E. gallinarum and E. casseliflavus. MBio. 3, e00318–e00311 (2012)Google Scholar
  15. 15.
    Huebner, J., Wang, Y., Krueger, W.A., Madoff, L.C., Martirosian, G., Boisot, S., Goldmann, D.A., Kasper, D.L., Tzianabos, A.O., Pier, G.B.: Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium. Infect. Immun. 67, 1213–1219 (1999)Google Scholar
  16. 16.
    Theilacker, C., Kaczynski, Z., Kropec, A., Fabretti, F., Sange, T., Holst, O., Huebner, J.: Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid. Infect. Immun. 74, 5703–5712 (2006)CrossRefGoogle Scholar
  17. 17.
    Theilacker, C., Kaczyński, Z., Kropec, A., Sava, I., Ye, L., Bychowska, A., Holst, O., Huebner, J.: Serodiversity of opsonic antibodies against Enterococcus faecalis--glycans of the cell wall revisited. PLoS One. 6, e17839 (2011)CrossRefGoogle Scholar
  18. 18.
    Qin, X., Galloway-Peña, J.R., Sillanpaa, J., Roh, J.H., Nallapareddy, S.R., Chowdhury, S., Bourgogne, A., Choudhury, T., Muzny, D.M., Buhay, C.J., Ding, Y., Dugan-Rocha, S., Liu, W., Kovar, C., Sodergren, E., Highlander, S., Petrosino, J.F., Worley, K.C., Gibbs, R.A., Weinstock, G.M., Murray, B.E.: Complete genome sequence of Enterococcus faecium strain TX16 and comparative genomic analysis of Enterococcus faecium genomes. BMC Microbiol. 12, 135 (2012)CrossRefGoogle Scholar
  19. 19.
    Laverde, D., Wobser, D., Romero-Saavedra, F., Hogendorf, W., van der Marel, G., Berthold, M., Kropec, A., Codee, J., Huebner, J.: Synthetic teichoic acid conjugate vaccine against nosocomial gram-positive bacteria. PLoS One. 9, e110953 (2014)CrossRefGoogle Scholar
  20. 20.
    Kodali, S., Vinogradov, E., Lin, F., Khoury, N., Hao, L., Pavliak, V., Jones, C.H., Laverde, D., Huebner, J., Jansen, K.U., Anderson, A.S., Donald, R.G.K.: A vaccine approach for the prevention of infections by multidrug-resistant Enterococcus faecium. J. Biol. Chem. 290, 19512–19526 (2015)CrossRefGoogle Scholar
  21. 21.
    van Schaik, W., Top, J., Riley, D.R., Boekhorst, J., Vrijenhoek, J.E.P., Schapendonk, C.M.E., Hendrickx, A.P.A., Nijman, I.J., Bonten, M.J.M., Tettelin, H., Willems, R.J.L.: Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island. BMC Genomics. 11, 239 (2010)CrossRefGoogle Scholar
  22. 22.
    Turula, V.E., Gore, T., Singh, S., Arumugham, R.G.: Automation of the anthrone assay for carbohydrate concentration determinations. Anal. Chem. 82, 1786–1792 (2010)CrossRefGoogle Scholar
  23. 23.
    Dische, Z., Shettles, L.B.: A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. Biol. Chem. 175, 595–603 (1948)Google Scholar
  24. 24.
    Lowry, O.H., Roberts, N.R., WU, M.L., Hixon, W.S., Crawford, E.J.: The quantitative histochemistry of brain. II. Enzyme measurements. J. Biol. Chem. 207, 19–37 (1954)Google Scholar
  25. 25.
    Kropec, A., Sava, I.G., Vonend, C., Sakinc, T., Grohmann, E., Huebner, J.: Identification of SagA as a novel vaccine target for the prevention of Enterococcus faecium infections. Microbiology. 157, 3429–3434 (2011)CrossRefGoogle Scholar
  26. 26.
    Haller, C., Berthold, M., Wobser, D., Kropec, A., Lauriola, M., Schlensak, C., Huebner, J.: Cell-wall glycolipid mutations and their effects on virulence of E. faecalis in a rat model of infective endocarditis. PLoS One. 9, e91863 (2014)CrossRefGoogle Scholar
  27. 27.
    Gründling, A., Schneewind, O.: Genes required for glycolipid synthesis and lipoteichoic acid anchoring in Staphylococcus aureus. J. Bacteriol. 189, 2521–2530 (2007)CrossRefGoogle Scholar
  28. 28.
    Romero-Saavedra, F., Laverde, D., Wobser, D., Michaux, C., Budin-Verneuil, A., Bernay, B., Benachour, A., Hartke, A., Huebner, J.: Identification of peptidoglycan-associated proteins as vaccine candidates for enterococcal infections. PLoS One. 9, e111880 (2014)CrossRefGoogle Scholar
  29. 29.
    Sava, I.G., Heikens, E., Huebner, J.: Pathogenesis and immunity in enterococcal infections. Clin. Microbiol. Infect. 16, 533–540 (2010)CrossRefGoogle Scholar
  30. 30.
    Wobser, D., Ali, L., Grohmann, E., Huebner, J., Sakinc, T.: A novel role for D-alanylation of lipoteichoic acid of enterococcus faecalis in urinary tract infection. PLoS One. 9, e107827 (2014)CrossRefGoogle Scholar
  31. 31.
    Theilacker, C., Sanchez-Carballo, P., Toma, I., Fabretti, F., Sava, I., Kropec, A., Holst, O., Huebner, J.: Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis. Mol. Microbiol. 71, 1055–1069 (2009)CrossRefGoogle Scholar
  32. 32.
    Diederich, A.-K., Wobser, D., Spiess, M., Sava, I.G., Huebner, J., Sakιnç, T.: Role of glycolipids in the pathogenesis of Enterococcus faecalis urinary tract infection. PLoS One. 9, e96295 (2014)CrossRefGoogle Scholar
  33. 33.
    Paganelli, F.L., Huebner, J., Singh, K.V., Zhang, X., van Schaik, W., Wobser, D., Braat, J.C., Murray, B.E., Bonten, M.J.M., Willems, R.J.L., Leavis, H.L.: Genome-wide screening identifies phosphotransferase system permease BepA to be involved in Enterococcus faecium endocarditis and biofilm formation. J. Infect. Dis. 214, 189–195 (2016)CrossRefGoogle Scholar
  34. 34.
    Fabretti, F., Theilacker, C., Baldassarri, L., Kaczynski, Z., Kropec, A., Holst, O., Huebner, J.: Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infect. Immun. 74, 4164–4171 (2006)CrossRefGoogle Scholar
  35. 35.
    Weidenmaier, C., Peschel, A., Xiong, Y.-Q., Kristian, S.A., Dietz, K., Yeaman, M.R., Bayer, A.S.: Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis. J. Infect. Dis. 191, 1771–1777 (2005)CrossRefGoogle Scholar
  36. 36.
    Neuhaus, F.C., Baddiley, J.: A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67, 686–723 (2003)CrossRefGoogle Scholar
  37. 37.
    Morath, S., Stadelmaier, A., Geyer, A., Schmidt, R.R., Hartung, T.: Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release. J. Exp. Med. 195, 1635–1640 (2002)CrossRefGoogle Scholar
  38. 38.
    Weintraub, A.: Immunology of bacterial polysaccharide antigens. Carbohydr. Res. 338, 2539–2547 (2003)CrossRefGoogle Scholar
  39. 39.
    Ada, G.: Vaccines and vaccination. N. Engl. J. Med. 345, 1042–1053 (2001)CrossRefGoogle Scholar
  40. 40.
    Weidenmaier, C., Peschel, A.: Teichoic acids and related cell-wall glycopolymers in gram-positive physiology and host interactions. Nat. Rev. Microbiol. 6, 276–287 (2008)CrossRefGoogle Scholar
  41. 41.
    Roberts, I.S.: The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu. Rev. Microbiol. 50, 285–315 (1996)CrossRefGoogle Scholar
  42. 42.
    Roy, D., Auger, J.-P., Segura, M., Fittipaldi, N., Takamatsu, D., Okura, M., Gottschalk, M.: Role of the capsular polysaccharide as a virulence factor for Streptococcus suis serotype 14. Can. J. Vet. Res. 79, 141–146 (2015)Google Scholar
  43. 43.
    Geiss-Liebisch, S., Rooijakkers, S.H.M., Beczala, A., Sanchez-Carballo, P., Kruszynska, K., Repp, C., Sakinc, T., Vinogradov, E., Holst, O., Huebner, J., Theilacker, C.: Secondary cell wall polymers of Enterococcus faecalis are critical for resistance to complement activation via mannose-binding lectin. J. Biol. Chem. 287, 37769–37777 (2012)CrossRefGoogle Scholar
  44. 44.
    Thurman, J.M., Holers, V.M.: The central role of the alternative complement pathway in human disease. J. Immunol. 176, 1305–1310 (2006)CrossRefGoogle Scholar
  45. 45.
    Kleine, B., Ali, L., Wobser, D., Sakιnç, T.: The N-terminal repeat and the ligand binding domain a of SdrI protein is involved in hydrophobicity of S. saprophyticus. Microbiol. Res. 172, 88–94 (2015)CrossRefGoogle Scholar
  46. 46.
    Nicolle, L.E.: Catheter associated urinary tract infections. Antimicrob Resist Infect Control. 3(23), (2014)Google Scholar
  47. 47.
    Shokoohizadeh, L., Mobarez, A.M., Zali, M.R., Ranjbar, R., Alebouyeh, M., Sakinc, T., Ali, L.: High frequency distribution of heterogeneous vancomycin resistant Enterococcous faecium (VREfm) in Iranian hospitals. Diagn. Pathol. 8(902), (2013)Google Scholar
  48. 48.
    Shankar, N., Lockatell, C.V., Baghdayan, A.S., Drachenberg, C., Gilmore, M.S., Johnson, D.E.: Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection. Infect. Immun. 69, 4366–4372 (2001)CrossRefGoogle Scholar
  49. 49.
    Kau, A.L., Martin, S.M., Lyon, W., Hayes, E., Caparon, M.G., Hultgren, S.J.: Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J mice. Infect. Immun. 73, 2461–2468 (2005)CrossRefGoogle Scholar
  50. 50.
    Wang, A., Athan, E., Pappas, P.A., Fowler, V.G., Olaison, L., Paré, C., Almirante, B., Muñoz, P., Rizzi, M., Naber, C., Logar, M., Tattevin, P., Iarussi, D.L., Selton-Suty, C., Jones, S.B., Casabé, J., Morris, A., Corey, G.R., Cabell, C.H.: International collaboration on endocarditis-prospective cohort study investigators: contemporary clinical profile and outcome of prosthetic valve endocarditis. JAMA. 297, 1354–1361 (2007)CrossRefGoogle Scholar
  51. 51.
    Donlan, R.M., Costerton, J.W.: Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15, 167–193 (2002)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Liaqat Ali
    • 1
    • 2
    Email author
  • Hubert E. Blum
    • 1
  • Türkân Sakιnç
    • 1
  1. 1.Division of Infectious Diseases, Department of Internal MedicineUniversity Medical Center FreiburgFreiburgGermany
  2. 2.Department of Biological SciencesNational University of Medical Sciences (NUMS)RawalpindiPakistan

Personalised recommendations