Abstract
To use a flow-based method to establish, quantify and visualize biofilms of Ureaplasma parvum. Absorbance readings of a U.parvum HPA5 culture were taken at 550nm every 3h for 30h 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 0·01mlmin-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 (0·599µm3 /µm2 ±0·152 and 4×108 CCU per ml, respectively) compared with static conditions (0·008µm3 /µm2 ±0·010 and no recoverable cells, respectively). SEM revealed pleomorphic cells, with signs of budding and possible membrane vesicle formation. Flow is an essential requirement for the establishment of U.parvum biofilms. 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.
Highlights
Mollicutes include several human and veterinary relevant pathogens
We present confocal scanning laser microscopy and scanning electron micrograph images of U. parvum which have been grown using this method
Ureaplasma parvum was grown under static conditions for 30 h with measurements of viability and media colour change as a marker for media alkalisation taken every 3 h (Fig. 1a)
Summary
Mollicutes include several human and veterinary relevant pathogens. These organisms differ from many bacteria by lacking a cell wall, being pleomorphic in shape, harbouring a genome with reduced coding capacity and an essential requirement for sterols. The bacteria within the genus Ureaplasma are unique among Mollicutes due to their sole utilization of urea in the acquisition of energy (Robertson et al 2002). Ureaplasmas colonize mucosal membranes within the genital tract of around 40–80% of women and 30% of men (Waites et al 2005). Colonization among asymptomatic individuals is common, several disease states have been associated. Ureaplasmas can ascend the female genital tract during pregnancy, leading to in utero infection, chorioamnionitis and subsequent
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