A requirement for flow to enable the development of Ureaplasma parvum biofilms in vitro

Abstract

Aims: To use a flow-based method to establish, quantify and visualize biofilms of Ureaplasma parvum.

Methods and results: Absorbance readings of a U. parvum HPA5 culture were taken at 550 nm every 3 h for 30 h in order to establish a growth curve, with viability determined by the number of colour changing units (CCUs). Biofilms were established using the DTU flow-cell with a flow rate of 0·01 ml min^-1 and compared to the static control. Titres of bacteria were determined by CCU and biofilm biomass was quantified by Syto9 staining and COMSTAT analysis. High-resolution images were obtained by scanning electron microscopy (SEM). Flow resulted in significantly more biofilm and higher cell titre (0·599 µm³ /µm² ± 0·152 and 4 × 10⁸ CCU per ml, respectively) compared with static conditions (0·008 µm³ /µm² ± 0·010 and no recoverable cells, respectively). SEM revealed pleomorphic cells, with signs of budding and possible membrane vesicle formation.

Conclusions: Flow is an essential requirement for the establishment of U. parvum biofilms.

Significance and impact of the study: This is the first quantification of biofilm biomass formed by U. parvum. It is now possible to establish viable biofilms of U. parvum which will allow for future testing of antimicrobial agents and understanding of virulence-associated with adhesion.

Keywords: Ureaplasma parvum; biofilm; flow cell; quantification; scanning electron microscopy.