Advertisement

Growing and Characterizing Biofilms Formed by Streptococcus pneumoniae

  • Yashuan Chao
  • Caroline Bergenfelz
  • Anders P. HakanssonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1968)

Abstract

It is estimated that over 80% of bacterial infections are associated with biofilm formation. Biofilms are organized bacterial communities formed on abiotic surfaces, such as implanted or inserted medical devices, or on biological surfaces, such as epithelial linings and mucosal surfaces. Biofilm growth is advantageous for the bacterial organism as it protects the bacteria from antimicrobial host factors and allows the bacteria to reside in the host without causing excessive inflammation. Like many other opportunistic pathogens of the respiratory tract, Streptococcus pneumoniae forms biofilms during asymptomatic carriage, which promotes, among other things, persistence in the niche, intraspecies and interspecies communication, and spread of bacterial DNA. Changes within the colonizing environment resulting from host assaults, such as virus infection, can induce biofilm dispersion where bacteria leave the biofilm and disseminate to other sites with ensuing infection. In this chapter, we present methodology to form complex biofilms in the nasopharynx of mice and to evaluate the biofilm structure and function in this environment. Furthermore, we present methods that recapitulate this biofilm phenotype in vitro by incorporating crucial factors associated with the host environment and describe how these models can be used to study biofilm function, transformation, and dispersion.

Key words

Biofilm Streptococcus pneumoniae Respiratory tract Nasopharynx Epithelium Mucosa Carriage Colonization Transformation Competence Dispersion Virulence 

References

  1. 1.
    Koo H, Falsetta ML, Klein MI (2013) The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm. J Dent Res 92:1065–1073CrossRefGoogle Scholar
  2. 2.
    Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK (2005) The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J Immunol 175:7512–7518CrossRefGoogle Scholar
  3. 3.
    Das J, Mokrzan E, Lakhani V, Rosas L, Jurcisek JA, Ray WC, Bakaletz LO (2017) Extracellular DNA and type IV pilus expression regulate the structure and kinetics of biofilm formation by nontypeable Haemophilus influenzae. MBio 8.  https://doi.org/10.1128/mBio.01466-17
  4. 4.
    Novotny LA, Jurcisek JA, Goodman SD, Bakaletz LO (2016) Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo. EBioMedicine 10:33–44CrossRefGoogle Scholar
  5. 5.
    Marks LR, Reddinger RM, Hakansson AP (2012) High levels of genetic recombination during nasopharyngeal carriage and biofilm formation in Streptococcus pneumoniae. MBio 3:e00200–e00212CrossRefGoogle Scholar
  6. 6.
    Wei H, Håvarstein LS (2012) Fratricide is essential for efficient gene transfer between pneumococci in biofilms. Appl Environ Microbiol 78:5897–5905CrossRefGoogle Scholar
  7. 7.
    Wolcott RD, Ehrlich GD (2008) Biofilms and chronic infections. JAMA 299:2682–2684CrossRefGoogle Scholar
  8. 8.
    Domenech M, García E, Prieto A, Moscoso M (2013) Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms. Environ Microbiol 15:502–516CrossRefGoogle Scholar
  9. 9.
    Hernandez-Jimenez E, Del Campo R, Toledano V, Vallejo-Cremades MT, Munoz A, Largo C, Arnalich F, Garcia-Rio F, Cubillos-Zapata C, Lopez-Collazo E (2013) Biofilm vs. planktonic bacterial mode of growth: which do human macrophages prefer? Biochem Biophys Res Commun 441:947–952CrossRefGoogle Scholar
  10. 10.
    Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322CrossRefGoogle Scholar
  11. 11.
    Arciola CR, Campoccia D, Montanaro L (2018) Implant infections: adhesion, biofilm formation and immune evasion. Nat Rev Microbiol 16:397–409CrossRefGoogle Scholar
  12. 12.
    Weimer KE, Armbruster CE, Juneau RA, Hong W, Pang B, Swords WE (2010) Coinfection with Haemophilus influenzae promotes pneumococcal biofilm formation during experimental otitis media and impedes the progression of pneumococcal disease. J Infect Dis 202:1068–1075CrossRefGoogle Scholar
  13. 13.
    Blanchette-Cain K, Hinojosa CA, Akula Suresh Babu R, Lizcano A, Gonzalez-Juarbe N, Munoz-Almagro C, Sanchez CJ, Bergman MA, Orihuela CJ (2013) Streptococcus pneumoniae biofilm formation is strain dependent, multifactorial, and associated with reduced invasiveness and immunoreactivity during colonization. MBio 4:e00745–e00713CrossRefGoogle Scholar
  14. 14.
    Marks LR, Davidson BA, Knight PR, Hakansson AP (2013) Interkingdom signaling induces Streptococcus pneumoniae biofilm dispersion and transition from asymptomatic colonization to disease. MBio 4:e00438–e00413CrossRefGoogle Scholar
  15. 15.
    Walsh RL, Camilli A (2011) Streptococcus pneumoniae is desiccation tolerant and infectious upon rehydration. MBio 2:e00092–e00011CrossRefGoogle Scholar
  16. 16.
    Marks LR, Reddinger RM, Hakansson AP (2014) Biofilm formation enhances fomite survival of Streptococcus pneumoniae and Streptococcus pyogenes. Infect Immun 82:1141–1146CrossRefGoogle Scholar
  17. 17.
    Bloomfield S, Exner M, Flemming HC, Goroncy-Bermes P, Hartemann P, Heeg P, Ilschner C, Krämer I, Merkens W, Oltmanns P, Rotter M, Rutala WA, Sonntag HG, Trautmann M (2015) Lesser-known or hidden reservoirs of infection and implications for adequate prevention strategies: where to look and what to look for. GMS Hyg Infect Control 10:Doc04PubMedPubMedCentralGoogle Scholar
  18. 18.
    Dettenkofer M, Block C (2005) Hospital disinfection: efficacy and safety issues. Curr Opin Infect Dis 18:320–325CrossRefGoogle Scholar
  19. 19.
    Chao Y, Bergenfelz C, Hakansson AP (2017) In vitro and in vivo biofilm formation by pathogenic streptococci. In: Nordenfelt P, Collin M (eds) Bacterial pathogens: methods and protocols, Methods in molecular biology, vol 1535. Humana Press, New York, pp 285–299CrossRefGoogle Scholar
  20. 20.
    Short KR, Diavatopoulos DA (2015) Chapter 15: Nasopharyngeal colonization with Streptococcus pneumoniae. In: Orihuela C, Hammerschmidt S, Brown J (eds) Streptococcus pneumoniae: molecular mechanisms of host-pathogen interactions. Elsevier, Academic Press, LondonGoogle Scholar
  21. 21.
    Dabernat H, Geslin P, Megraud F, Begue P, Boulesteix J, Dubreuil C, de La Roque F, Trinh A, Scheimberg A (1998) Effects of cefixime or co-amoxiclav treatment on nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae in children with acute otitis media. J Antimicrob Chemother 41:253–258CrossRefGoogle Scholar
  22. 22.
    Dagan R, Leibovitz E, Greenberg D, Yagupsky P, Fliss DM, Leiberman A (1998) Dynamics of pneumococcal nasopharyngeal colonization during the first days of antibiotic treatment in pediatric patients. Pediatr Infect Dis J 17:880–885CrossRefGoogle Scholar
  23. 23.
    Marks LR, Parameswaran GI, Hakansson AP (2012) Pneumococcal interactions with epithelial cells are crucial for optimal biofilm formation and colonization in vitro and in vivo. Infect Immun 80:2744–2760CrossRefGoogle Scholar
  24. 24.
    Ehrlich GD, Veeh R, Wang X, Costerton JW, Hayes JD, Hu FZ, Daigle BJ, Ehrlich MD, Post JC (2002) Mucosal biofilm formation on middle-ear mucosa in the chinchilla model of otitis media. JAMA 287:1710–1715CrossRefGoogle Scholar
  25. 25.
    Sanderson AR, Leid JG, Hunsaker D (2006) Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis. Laryngoscope 116:1121–1126CrossRefGoogle Scholar
  26. 26.
    Hall-Stoodley L, Hu FZ, Gieseke A, Nistico L, Nguyen D, Hayes J, Forbes M, Greenberg DP, Dice B, Burrows A, Wackym PA, Stoodley P, Post JC, Ehrlich GD, Kerschner JE (2006) Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA 296:202–211CrossRefGoogle Scholar
  27. 27.
    Reid SD, Hong W, Dew KE, Winn DR, Pang B, Watt J, Glover DT, Hollingshead SK, Swords WE (2009) Streptococcus pneumoniae forms surface-attached communities in the middle ear of experimentally infected chinchillas. J Infect Dis 199:786–794CrossRefGoogle Scholar
  28. 28.
    Charalambous BM, Leung MH (2012) Pneumococcal sepsis and nasopharyngeal carriage. Curr Opin Pulm Med 18:222–227CrossRefGoogle Scholar
  29. 29.
    Shak JR, Vidal JE, Klugman KP (2013) Influence of bacterial interactions on pneumococcal colonization of the nasopharynx. Trends Microbiol 21:129–135CrossRefGoogle Scholar
  30. 30.
    Gilley RP, Orihuela CJ (2014) Pneumococci in biofilms are non-invasive: implications on nasopharyngeal colonization. Front Cell Infect Microbiol 4:163CrossRefGoogle Scholar
  31. 31.
    Chao Y, Marks LR, Pettigrew MM, Hakansson AP (2015) Streptococcus pneumoniae biofilm formation and dispersion during colonization and disease. Front Cell Infect Microbiol 4(194):1–16Google Scholar
  32. 32.
    Allegrucci M, Hu FZ, Shen K, Hayes J, Ehrlich GD, Post JC, Sauer K (2006) Phenotypic characterization of Streptococcus pneumoniae biofilm development. J Bacteriol 188:2325–2335CrossRefGoogle Scholar
  33. 33.
    Moscoso M, Garcia E, Lopez R (2006) Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J Bacteriol 188:7785–7795CrossRefGoogle Scholar
  34. 34.
    Munoz-Elias EJ, Marcano J, Camilli A (2008) Isolation of Streptococcus pneumoniae biofilm mutants and their characterization during nasopharyngeal colonization. Infect Immun 76:5049–5061CrossRefGoogle Scholar
  35. 35.
    Sanchez CJ, Shivshankar P, Stol K, Trakhtenbroit S, Sullam PM, Sauer K, Hermans PW, Orihuela CJ (2010) The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms. PLoS Pathog 6:e1001044CrossRefGoogle Scholar
  36. 36.
    Yadav MK, Kwon SK, Cho CG, Park SW, Chae SW, Song JJ (2012) Gene expression profile of early in vitro biofilms of Streptococcus pneumoniae. Microbiol Immunol 56:621–629CrossRefGoogle Scholar
  37. 37.
    Sanchez CJ, Kumar N, Lizcano A, Shivshankar P, Dunning Hotopp JC, Jorgensen JH, Tettelin H, Orihuela CJ (2011) Streptococcus pneumoniae in biofilms are unable to cause invasive disease due to altered virulence determinant production. PLoS One 6:e28738CrossRefGoogle Scholar
  38. 38.
    Keck T, Leiacker R, Riechelmann H, Rettinger G (2000) Temperature profile in the nasal cavity. Laryngoscope 110:651–654CrossRefGoogle Scholar
  39. 39.
    Fux CA, Costerton JW, Stewart PS, Stoodley P (2005) Survival strategies of infectious biofilms. Trends Microbiol 13:34–40CrossRefGoogle Scholar
  40. 40.
    Chonmaitree T, Howie VM, Truant AL (1986) Presence of respiratory viruses in middle ear fluids and nasal wash specimens from children with acute otitis media. Pediatrics 77:698–702PubMedGoogle Scholar
  41. 41.
    Diavatopoulos DA, Short KR, Price JT, Wilksch JJ, Brown LE, Briles DE, Strugnell RA, Wijburg OL (2010) Influenza A virus facilitates Streptococcus pneumoniae transmission and disease. FASEB J 24(6):1789–1798CrossRefGoogle Scholar
  42. 42.
    Pettigrew MM, Gent JF, Pyles RB, Miller AL, Nokso-Koivisto J, Chonmaitree T (2011) Viral-bacterial interactions and risk of acute otitis media complicating upper respiratory tract infection. J Clin Microbiol 49:3750–3755CrossRefGoogle Scholar
  43. 43.
    Pettigrew MM, Marks LR, Kong Y, Gent JF, Roche-Hakansson H, Hakansson AP (2014) Dynamic changes in the Streptococcus pneumoniae transcriptome during transition from biofilm formation to invasive disease upon influenza A virus infection. Infect Immun 82:4607–4619CrossRefGoogle Scholar
  44. 44.
    Briles DE, Novak L, Hotomi M, van Ginkel FW, King J (2005) Nasal colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci. Infect Immun 73:6945–6951CrossRefGoogle Scholar
  45. 45.
    Kadioglu A, Weiser JN, Paton JC, Andrew PW (2008) The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 6:288–301CrossRefGoogle Scholar
  46. 46.
    Lewis K (2010) Persister cells. Annu Rev Microbiol 64:357–372CrossRefGoogle Scholar
  47. 47.
    Fux CA, Stoodley P, Hall-Stoodley L, Costerton JW (2003) Bacterial biofilms: a diagnostic and therapeutic challenge. Expert Rev Anti-Infect Ther 1:667–683CrossRefGoogle Scholar
  48. 48.
    Oggioni MR, Trappetti C, Kadioglu A, Cassone M, Iannelli F, Ricci S, Andrew PW, Pozzi G (2006) Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Mol Microbiol 61:1196–1210CrossRefGoogle Scholar
  49. 49.
    Vidal JE, Howery KE, Ludewick HP, Nava P, Klugman KP (2013) Quorum-sensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells. Infect Immun 81:1341–1353CrossRefGoogle Scholar
  50. 50.
    Wu HY, Nguyen HH, Russell MW (1997) Nasal lymphoid tissue (NALT) as a mucosal immune inductive site. Scand J Immunol 46:506–513CrossRefGoogle Scholar
  51. 51.
    Smith AW, Roche H, Trombe MC, Briles DE, Hakansson A (2002) Characterization of the dihydrolipoamide dehydrogenase from Streptococcus pneumoniae and its role in pneumococcal infection. Mol Microbiol 44:431–448CrossRefGoogle Scholar
  52. 52.
    Hakenbeck R, Briese T, Chalkley L, Ellerbrok H, Kalliokoski R, Latorre C, Leinonen M, Martin C (1991) Antigenic variation of penicillin-binding proteins from penicillin-resistant clinical strains of Streptococcus pneumoniae. J Infect Dis 164:313–319CrossRefGoogle Scholar
  53. 53.
    Rieger M, Denapaite D, Bruckner R, Maurer P, Hakenbeck R (2017) Draft genome sequences of two Streptococcus pneumoniae serotype 19A sequence Type 226 clinical isolates from Hungary, Hu17 with high-level beta-lactam resistance and Hu15 of a penicillin-sensitive phenotype. Genome Announc 5(20):e00401–e00417CrossRefGoogle Scholar
  54. 54.
    van de Rijn I, Kessler RE (1980) Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun 27:444–448PubMedPubMedCentralGoogle Scholar
  55. 55.
    Hammerschmidt S, Wolff S, Hocke A, Rosseau S, Muller E, Rohde M (2005) Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells. Infect Immun 73:4653–4667CrossRefGoogle Scholar
  56. 56.
    Carmen JC, Nelson JL, Beckstead BL, Runyan CM, Robison RA, Schaalje GB, Pitt WG (2004) Ultrasonic-enhanced gentamicin transport through colony biofilms of Pseudomonas aeruginosa and Escherichia coli. J Infect Chemother 10:193–199CrossRefGoogle Scholar
  57. 57.
    Abdi-Ali A, Mohammadi-Mehr M, Agha Alaei Y (2006) Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa. Int J Antimicrob Agents 27:196–200CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Yashuan Chao
    • 1
  • Caroline Bergenfelz
    • 1
  • Anders P. Hakansson
    • 1
    Email author
  1. 1.Wallenberg Laboratory, Division of Experimental Infection Medicine, Department of Translational MedicineLund UniversityMalmöSweden

Personalised recommendations