Visualization of invivo biofilm developing on silicone implants and the interaction with the cellular response
Abstract number: P1901
van Gennip M., Christensen L., Qvortrup K., Alhede M., Jensen P., Høiby N., Givskov M., Bjarnsholt T.
Objective:Pseudomonas aeruginosa is associated with infections of medical implants in humans, where they form biofilms. The biofilm mode of growth contributes to the tolerance against treatment with antibiotics as well as to the host response. Polymorphonuclear leukocytes (PMNs), which are part of the innate immunity and some of the first cell types to be recruited to the site of infection, most often fail to eradicate the biofilm infection. P. aeruginosa produces rhamnolipids which causes the PMNs to undergo a necrotic death. We have previously shown that a P. aeruginosa mutant unable to produce rhamnolipids was cleared from flat implants inserted into the peritoneal cavity of mice. By modifying the in vivo foreign-body infection model established by Christensen et al. in 2007 using 4 mm silicone tubes (id 4 mm, od 6 mm) instead of flat, square implants, we followed the development of P. aeruginosa biofilms as well as the pathogen-host interaction by means of quantitative bacteriology and scanning electron microscopy (SEM).
Methods: Wild-type (WT) P. aeruginosa (PAO1) and its corresponding rhlA mutant were harvested by centrifugation from an overnight culture and resuspended in 0.9% NaCl to an OD600nm of 0.1. The implant tubes were colonized for 20-hours with shaking at 110 rpm and inserted in the peritoneal cavity of BALB/c mice at day 0. Tubes removed from euthanized mice were either prepared for SEM (fixed in 2% glutaraldehyde, post-fixed in 1% OsO4, critical point dried using CO2 and sputter coated with gold according to standard procedures) or evaluated for quantitative bacteriology (CFU per implant). Specimens for SEM were investigated with a FEI XL Feg30 SEM microscope.
Results: Quantitative bacteriology showed that the mice were able to clear the rhlA mutant by day 3, but the WT was still cultivated on day 7 post-insertion. The SEM pictures revealed that by day 1 a biofilm had formed, and subsequently a continuously increase in bacterial biomass. As for the host response it was evident that by day 1 an abundant number of PMNs had been attracted and seemed to enter the tubes from the tube openings. Even more PMNs were seen on day 2, however, most of them seemed to be damaged and embedded in the biofilm matrix.
Conclusion: Quantitative bacteriology confirmed our previous findings that a rhlA mutant is cleared faster than the WT. The SEM visualization confirmed this finding and clearly emphasised the WT enabled destruction of the PMNs.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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