[go: up one dir, main page]
Skip to main content
Springer Nature Link
Log in

Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy

  • Applied Microbial and Cell Physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200–900 cm−1 region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm−1) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Amano KL, Fukushi K, Watanabe M (1990) Biochemical and immunological comparison of lipopolysaccharides from Bordetella species. J Gen Microbiol 136:481–487

    Article  CAS  PubMed  Google Scholar 

  • Batsoulis AN, Nacos MK, Pappas CS, Tarantilis PA, Mavromoustakos T, Polissiou MG (2004) Determination of uronic acids in isolated hemicelluloses from kenaf using diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and the curve-fitting deconvolution method. Appl Spectrosc 58:199–202

    Article  CAS  PubMed  Google Scholar 

  • Beech I, Hanjagsit L, Kalaji M, Neal AL, Zinkevich V (1999) Chemical and structural characterization of exopolymers produced by Pseudomonas sp. NCIMB 2021 in continuous culture. Microbiology 145:1491–1497

    Article  CAS  PubMed  Google Scholar 

  • Belcher CE, Drenkow J, Kehoe B, Gingeras TR, McNamara N, Lemjabbar H, Basbaum C, Relman DA (2000) The transcriptional responses of respiratory epithelial cells to Bordetella pertussis reveal host defensive and pathogen counter-defensive strategies. Proc Natl Acad Sci U S A 97:13847–13852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54:484–489

    Article  CAS  PubMed  Google Scholar 

  • Bosch A, Massa NE, Donolo AS, Yantorno OM (2000) Molecular characterisation by infrared spectroscopy of Bordetella pertussis growing in biofilm. Phys Status Solidi 220:635–640

    Article  CAS  Google Scholar 

  • Brown M, Barker J (1999) Unexplored reservoirs of pathogenic bacteria: protozoa and biofilms. Trends Microbiol 7:46–50

    Article  CAS  PubMed  Google Scholar 

  • Brown MR, Williams P (1985) The influence of environment on envelope properties affecting survival of bacteria in infections. Annu Rev Microbiol 39:527–556

    Article  CAS  PubMed  Google Scholar 

  • Cherry JD, Brunell PA, Golden GS, Karzon DT (1988) Report of the task force on pertussis and pertussis immunization. Pediatrics 81:939–984

    Article  Google Scholar 

  • Chung JY, Wilkie I, Boyce JD, Townsend KM, Frost AJ, Ghoddusi M, Adler B (2001) Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A. Infect Immun 69:2487–2492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Coote JG (1991) Antigenic switching and pathogenicity: environmental effects on virulence gene expression in Bordetella pertussis. J Gen Microbiol 137:2493–2503

    Article  CAS  PubMed  Google Scholar 

  • Costerton JW (1985) The role of bacterial exopolysaccharides in nature and disease. Dev Ind Microbiol 26:249–261

    CAS  Google Scholar 

  • Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marie TJ (1987) Bacterial biofilms in nature and disease. Annu Rev Microbiol 41:435–464

    Article  CAS  PubMed  Google Scholar 

  • Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM (1995) Microbial biofilms. Annu Rev Microbiol 711–745

    Article  CAS  PubMed  Google Scholar 

  • Costerton JW, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlich G (2003) The application of biofilm science to the study and control of chronic bacterial infections. J Clin Invest 112:1466–1477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Davies DG, Chakrabarty AM, Geesey GG (1993) Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa. Appl Environ Microbiol 59:1181–1186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • De Kievit TR, Gillis R, Marx S, Brown C, Iglewski BH (2001) Quorum-sensing genes in Pseudomonas aeruginosa biofilms: their role and expression patterns. Appl Environ Microbiol 67:1865–1873

    Article  PubMed  PubMed Central  Google Scholar 

  • Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881–890

    Article  PubMed  PubMed Central  Google Scholar 

  • Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Doyle RJ, Rosenberg M (1995) Adhesion of microbial pathogens. In: Doyle RJ, Ofek I (eds) Methods in enzymology, vol 253. Academic, San Diego, pp 542–550

    Google Scholar 

  • Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

    Article  CAS  Google Scholar 

  • Evans E, Brown MRW,Gilbert P (1994) Iron chelator, exopolysaccharide and protease production in Staphylococcus epidermidis: a comparative study of the effects of specific growth rate in biofilm and planktonic culture. Microbiology 140:153–157

    Article  CAS  PubMed  Google Scholar 

  • Fett WF, Wells JM, Cescutti P, Wijey C (1995) Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production. Appl Environ Microbiol 61:513–517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Figueroa LA, Silverstein JA (1989) Ruthenium red adsorption method for measurement of extracellular polysaccharides in sludge flocs. Biotechnol Bioeng 33:941–947

    Article  CAS  PubMed  Google Scholar 

  • Flemming H-C, Wingender J, Griegbe T, Mayer C (2000) Physico-chemical properties of biofilms. In: Evans LV (ed) Biofilm: recent advances in their study and control. Harwood, Amsterdam, pp 19–34

    Chapter  Google Scholar 

  • Graeff-Wohlleben H, Deppisch H, Gross R (1995) Global regulatory mechanisms affect virulence gene expression in Bordetella pertussis. Mol Gen Genet 247:86–94

    Article  CAS  PubMed  Google Scholar 

  • Grube M, Zagreba E, Gromozova E, Fomina M (1999) Comparative investigation of the macromolecular composition of mycelia forms Thielavia terrestris by infrared spectroscopy. Vibr Spectrosc 19:301–306

    Article  CAS  Google Scholar 

  • Helm D, Naumann D (1995) Identification of some bacteria cell components by FT-IR spectroscopy. FEMS Microbiol Lett 126:75–80

    Article  CAS  Google Scholar 

  • Helm D, Labischinsky H, Schallehn G, Naumann D (1991) Classification and identification of bacteria by Fourier-transform infrared spectroscopy. J Gen Microbiol 137:69–79

    CAS  PubMed  Google Scholar 

  • Hitchcock P, Brown T (1983) Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154:269–277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hozbor D, Rodríguez ME, Samo A, Lagares A, Yantorno OM (1993) Release of lipopolysaccharide during Bordetella pertussis growth. Res Microbiol 144:201–209

    Article  CAS  PubMed  Google Scholar 

  • Kacuráková M, Capek P, Sasinková V, Wellner N, Ebringerová A (2000) FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydr Polym 43:195–203

    Article  Google Scholar 

  • Khelef N, Bachelet C-M, Vargaftig BB, Guiso N (1994) Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins. Infect Immun 62:2893–2900

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 117:680–685

    Article  Google Scholar 

  • Lesse AJ, Campagnari AA, Bittner WE, Apicella MA (1990) Increased resolution of lipopolysaccharides and lipoligosaccharides utilising tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Immunol Methods 126:109–117

    Article  CAS  PubMed  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Article  CAS  PubMed  Google Scholar 

  • Maagd R, Lugtenberg B (1986) Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol 167:1083–1085

    Article  PubMed  PubMed Central  Google Scholar 

  • Martínez de Tejeda G, Miller JF, Cotter PA (1996) Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica. Mol Microbiol 22:895–908

    Article  Google Scholar 

  • Mayer C, Moritz R, Kirschner C, Borchard W, Maibaum R, Wingender J, Flemming H-C (1999) The role of intermolecular interactions: studies on model systems for bacterial biofilms. Int J Biol Macromol 26:3–16

    Article  CAS  PubMed  Google Scholar 

  • Meluleni G, Grout JM, Evans DJ, Pier GB (1995) Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients. J Immunol 155:2029–2038

    CAS  PubMed  Google Scholar 

  • Mooi FR, van Loo IHM, King AJ (2001) Adaptation of Bordetella pertussis to vaccination: a cause of its reemergence? Emerg Infect Dis 7:526–528

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moreno J, Vargas MA, Madiedo JM, Muños J, Rivas J, Guerrero MG (2000) Chemical and rheological properties of an extracellular polysaccharide produced by the cyanobacterium Anabaena sp. ATCC 33047. Biotechnol Bioeng 67:283–290

    Article  CAS  PubMed  Google Scholar 

  • Naumann D (2000) Infrared spectroscopy in microbiology. Wiley, Chichester

    Google Scholar 

  • Naumann D, Helm D, Labischinski H (1991) Microbiological characterizations by FT-IR spectroscopy. Nature 351:81–82

    Article  CAS  PubMed  Google Scholar 

  • Navarini L, Stredansky M, Matulova M, Bertocchi C (1997) Production and characterization of an exopolysaccharide from Rhizobium hedysari HCNT1. Biotechnol Lett 19:231–1234

    Article  Google Scholar 

  • Nebenzahl YM, Porat N, Lifshitz S, Novick S, Levi A, Ling E, Liron O, Mordechai S, Sahu RK, Dagan R (2004) Virulence of Streptoccoccus pneumoniae may be determined independently of capsular polysaccharide. FEMS Microbiol Lett 233:147–152

    Article  CAS  Google Scholar 

  • Nichols P, Henson J, Guckert J, Nivens DE, White DC (1985) Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria–polymer mixtures and biofilms. J Microbiol Methods 4:79–94

    Article  CAS  PubMed  Google Scholar 

  • Nivens DE, Palmer JR Jr, White DC (1995) Continuous nondestructive monitoring of microbial biofilms: a review of analytical techniques. J Ind Microbiol 15:263–276

    Article  CAS  Google Scholar 

  • Nivens DE, Ohman DE, Williams J, Franklin M (2001) Role of alginate and its O acetylation in formation of Pseudomonas aeurginosa microcolonies and biofilms. J Bacteriol 183:1047–1057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pace JL, Chai T, Rossi HA, Jiang X (1997) Effect of bile on Vibrio parahaemolyticus. Appl Environ Microbiol 63:2372–2377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pelkonen S, Hayrinen J, Finne J (1988) Polyacrylamide gel electrophoresis of the capsular polysaccharide of Escherichia coli K1 and other bacteria. J Bacteriol 170:2646–2653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peppler MS (1984) Two physically and serologically distinct lipopolysaccharide profiles in strains of Bordetella pertussis and their phenotype variants. Infect Immun 43:224–232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Prigent-Combaret C, Vidal O, Dorel C, Lejeune P (1999) Abiotic surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli. J Bacteriol 181:5993–6002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qadri F, Haque MdA, Hossain A, Albert MJ (1994) Production of slime polysaccharides by Shigella dysenteriae type 1. Microbiol Immunol 38:11–18

    Article  CAS  PubMed  Google Scholar 

  • Qiushui H, Makinen J, Berbers G, Mooi FR, Viljanesn MK, Arvilommi H, Mertsola J (2003) Bordetella pertussis protein pertactin induces type-specific antibodies: one possible explanation for the emergence of antigenic variants? J Infect Dis 187:1200–1205

    Article  Google Scholar 

  • Rodríguez ME, Hozbor DF, Samo AL, Ertola R, Yantorno OM (1994) Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide of Bordetella pertussis. J Ind Microbiol 13:273–278

    Article  Google Scholar 

  • Schmitt J, Flemming H-C (1998) FTIR-spectroscopy in microbial and material analysis. Int Biodeterior Biodegrad 41:1–11

    Article  CAS  Google Scholar 

  • Schmitt J, Nivens D, White DC, Flemming H-C (1995) Changes of biofilm properties in response to sorbed substances—an FT-IR/ATR study. Water Sci Technol 32:S149–S155

    Article  Google Scholar 

  • Shiau AL, Wu CL (1998) The inhibitory effect of Staphylococcus epidermidis slime on the phagocytosis of murine peritoneal macrophages is interferon-independent. Microbiol Immunol 42:33–40

    Article  CAS  PubMed  Google Scholar 

  • Stainer DW, Scholte MJ (1971) A simple chemically defined medium for production of phase I Bordetella pertussis. J Gen Microbiol 63:211–220

    Article  Google Scholar 

  • Sutherland IW (1997) Bacterial exopolysaccharides—their nature and production. In: Sutherland IW (ed) Surface carbohydrates of the prokaryotic cell. Academic, London, pp 27–96

    Google Scholar 

  • Sutherland IW (2001) Biofilm exopolysaccharides: a strong and sticky framework. Microbiology 147:3–9

    Article  CAS  PubMed  Google Scholar 

  • Synytsya A, Copíková J, Matejka P, Machovic V (2003) Fourier transform Raman and infrared spectroscopy of pectins. Carbohydr Polym 54:97–106

    Article  CAS  Google Scholar 

  • Uhlinger DJ, White DC (1983) Relationship between physiological status and formation of extracellular polysaccharide glycocalyx, in Pseudomonas atlantica. Appl Environ Microbiol 45:64–70

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Valverde C, Hozbor DF, Lagares A (1997) Rapid preparation of affinity-purified lipopolysaccharide samples for electrophoretic analysis. BioTechniques 22:230–236

    Article  CAS  PubMed  Google Scholar 

  • Vandevivere P, Kirchman DL (1993) Attachment stimulates exopolysaccharide synthesis by a bacterium. Appl Environ Microbiol 59:3280–3286

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • van der Mei H, Noordmans J, Busscher HJ (1989) Physicochemical surface properties of nonencapsulated and encapsulated coagulase-negative Staphylococci. Appl Environ Microbiol 55:2806–2814

    Article  PubMed  PubMed Central  Google Scholar 

  • van Loosdrecht MCM, Lyklema J, Norde W, Schraa G, Zehnder AJB (1990) The role of bacterial cell wall hydrophobicity in adhesion. Appl Environ Microbiol. 53:1893–1897

    Article  Google Scholar 

  • van Rie A, Hethcote HW (2004) Adolescent and adult pertussis vaccination: computer simulations of five new strategies. Vaccine 22:3154–3165

    Article  PubMed  Google Scholar 

  • Waller LN, Fox N, Fox KF, Fox A, Price RL (2004) Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis. J Microbiol Methods 58:23–30

    Article  CAS  PubMed  Google Scholar 

  • Weiser J, Bae ND, Epino H, Gordon SB, Kapoor M, Zenewcz LA, Shchepetov M (2001) Changes in availability of oxygen accentuate differences in capsular polysaccharide expression by phenotypic variants and clinical isolates of Streptococcus pneumoniae. Infect Immun 69:5430–5439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yasuda H, Ajiki Y, Aoyama J, Yokota T (1994) Interaction between human polymorphonuclear leucocytes and bacteria released from in-vitro bacterial biofilm models. J Med Microbiol 41:359–367

    Article  CAS  PubMed  Google Scholar 

  • Zeroual W, Choisy C, Doglia SM, Bobichon H, Angiboust J-F, Manfait M (1994) Monitoring of bacterial growth and structural analysis as probed by FT-IR spectroscopy. Biochim Biophys Acta 1222:171–178

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from FONCYT, PICT 98-06-03824. A. Bosch is a member of the CIC Provincia de Buenos Aires, D. Serra is a recipient of a fellowship from CONICET, and C. Prieto is a recipient from FOMEC. We are grateful to M.E. Rodríguez for providing us B. pertussis cells grown in continuous culture. We thank J. Figari for his excellent technical assistance.

Author information

Authors and Affiliations

  1. Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), Facultad de Ciencias Exactas, UNLP, calle 50 y 115, 1900, La Plata, Argentina

    A. Bosch, D. Serra, C. Prieto & O. Yantorno

  2. Synthon GmbH, Im, Neuenheimer Feld 583, 69120, Heidelberg, Germany

    J. Schmitt

  3. Robert-Koch-Institut, P13, Nordufer 20, 13353, Berlin, Germany

    D. Naumann

Authors
  1. A. Bosch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Serra
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Prieto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Schmitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D. Naumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. O. Yantorno
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Schmitt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bosch, A., Serra, D., Prieto, C. et al. Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy. Appl Microbiol Biotechnol 71, 736–747 (2006). https://doi.org/10.1007/s00253-005-0202-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-005-0202-8

Keywords