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-μm 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 μm in diameter and 24 μm 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.

Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis

“…Recent studies show that biofilm formation in the host respiratory tract is important for adaptation and survival of B. pertussis during an infection (Serra et al, 2007(Serra et al, , 2008. Here, we demonstrated that in B. pertussis, (p)ppGpp was synthesized by RelA and SpoT, and that these (p)ppGpp had an important role in B. pertussis biofilm formation.…”

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis

“…In Bordetella, BvgA, together with the sensor protein BvgS, integrates the BvgAS two-component system, which controls the expression of a complex array of virulence factors through a phosphorylation cascade in response to environmental stimuli [50]. The recent finding that the BvgAS system is involved in regulating biofilm formation [2,4,20] together with the results presented in this work, raise intriguing questions about the virulence phenotype of the sessile cells. Another interesting observation, which requires further attention, is the increased expression of Sadenosylmethionine synthetase in biofilm.…”

“…Although historically the occurrence of pertussis was associated with an acute infection, primarily in not-or under-immunized infants, a shift in the incidence of the disease towards adolescents and adults with the appearance of persistent asymptomatic or mild infections is becoming increasingly evident [1]. Recent reports support the hypothesis that B. pertussis could adopt a biofilm lifestyle as a strategy to colonize the host, but the mechanisms leading to persistent B. pertussis infections are largely unknown [2][3][4]. Biofilm is a community-based mode of existence of microbes in which cells are enclosed in a self-producing matrix and adhered to an inert or living surface [5].…”

“…In B. pertussis physiologic and pathogenic aspects have been extensively studied focusing on the planktonic mode of growth, while only a few works so far have considered the biofilm lifestyle of this bacterial pathogen [2,[20][21][22]. Therefore, generally only limited data is available on the nature of B. pertussis biofilm and information on a proteomic level is completely missing.…”

Fourier Transform Infrared Spectroscopy for Rapid Identification of Nonfermenting Gram-Negative Bacteria Isolated from Sputum Samples from Cystic Fibrosis Patients

“…The IR spectra of intact bacteria provide highly specific patterns that allow microbial cells to be distinguished at different taxonomic levels and have frequently been employed for the rapid and accurate identification of microorganisms even to the strain level (24,29,35,45). FTIR is easy to implement, allows the analysis of small quantities of biomass, and requires no consumables or reagents (38,52). Standardization of the cultivation conditions and the sampling and measurement parameters enabled the creation of reference libraries containing spectra for well-identified microbes.…”