title: Continuous nondestructive monitoring of Bordetella pertussis biofilms by Fourier transform infrared spectroscopy and other corroborative techniques.
abstract: This work describes the application of several analytical techniques to characterize the development of Bordetella pertussis biofilms and to examine, in particular, the contribution of virulence factors in this development. Growth of surface-attached virulent and avirulent B. pertussis strains was monitored in continuous-flow chambers by techniques such as the crystal violet method, and nondestructive methodologies like fluorescence microscopy and Fourier transform (FT) IR spectroscopy. Additionally, B. pertussis virulent and avirulent strains expressing green fluorescent protein were grown adhered to the base of a glass chamber of 1-microm thickness. Three-dimensional images of mature biofilms, acquired by confocal laser scanning microscopy, were quantitatively analysed by means of the computer program COMSTAT. Our results indicate that only the virulent (Bvg(+)) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm. In the virulent phase these bacteria are capable of producing a biofilm consisting of microcolonies of approximately 200 microm in diameter and 24 microm in depth. FTIR spectroscopy allowed us not only to follow the dynamics of biofilm growth through specific biomass and biofilm marker absorption bands, but also to monitor the maturation of the biofilm by means of the increase of the carbohydrate-to-protein ratio.
authors.affiliation: Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI, CONICET), Facultad de Ciencias Exactas, UNLP, Calles 50 y 115, 1900 La Plata, Argentina.
authors.forename: Diego
authors.initials: D
authors.lastname: Serra
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bosch
authors.forename: Daniela M
authors.initials: DM
authors.lastname: Russo
authors.forename: María E
authors.initials: ME
authors.lastname: Rodríguez
authors.forename: Angeles
authors.initials: A
authors.lastname: Zorreguieta
authors.forename: Juergen
authors.initials: J
authors.lastname: Schmitt
authors.forename: Dieter
authors.initials: D
authors.lastname: Naumann
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Yantorno
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D001886:Bordetella pertussis / Q000737:chemistry / Q000166:cytology* / Q000302:isolation & purification / Q000502:physiology*; D049109:Cell Proliferation; D015169:Colony Count, Microbial / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*