M037: QUANTITATIVE METHODS TO EVALUATE PSEUDOMONAS AERUGINOSA
ADHERENCE TO RESPIRATORY EPITHELIUM.
S.Girod de Bentzmann. Roger P. Bonnet N., Puchelle E. (INSERM
U314, CHR Maison Blanche,45 rue Cognacq Jay,51092 Reims, France).
The affinity of Pseudomonas aeruginosa (PA) for human respiratory epithelium is largely dependent on the cellular phenotype. We have previously demonstrated that damaged and repairing cells exhibit a high affinity for PA, whereas PA does not adhere to well differentiated cells ciliated and non ciliated cells (Girod de Bentzmann et al., Infect. Immun., 1994, 62, 704). We studied the adherence of PA to human respiratory epithelium in primary culture with scanning electron microscopy (SEM) and consequently developed quantitative methods to evaluate PA adherence in SEM. PA is able to adhere to human respiratory epithelial cells, as isolated bacteria as well as aggregates. These aggregates have been reported to be constituted by PA and secretory products from respiratory epithelial cells such as fibronectin and mucins (Plotkowski et al., J. Clin. Invest., 1991 87, 2018). We firstly evaluated the filling rate of PA aggregates with classical stereological methods performed on 2D sections of bacterial aggregates observed in TEM. With the help of an image analyzer, we superimposed stereological grids which consisted of a lattice of points whose periodicity varied as a function of magnification to TEM images of PA aggregates. The points were counted in the PA aggregates and in the bacteria themselves. We also evaluated the surface of the total aggregate and the surface occupied by PA within the aggregate and found a close correlation between these two parameters(r=0.99, p<0.0001). Whatever the size of bacterial aggregate, we found that only 50% of the aggregate was occupied by PA. Quantitation of PA adherence to human respiratory epithelium in primary culture was performed on a Philips SEM XL 30 coupled to an image analyzer at a magnification Df 2,700 and a voltage of 10kV. The quantitation consisted of determining the surface of each cellular phenotype, counting isolated PA and determining the surface of PA aggregates. Taking into account the previous results, the software that we developed was able to report the total number of adherent PA, the number of isolated PA and the number of PA in aggregates per square micrometer of epithelial surface. These quantitative methods are particularly relevant for evaluating PA adherence to the different phenotypes of respiratory epithelial cells, and to study the effect of molecules able to prevent PA adherence.
This work was supported by the Association Francaise de Lutte contre la Mucoviscidose (AFLM).