Real-time PCR method for the quantification of Burkholderia cepacia complex attached to lung epithelial cells and inhibition of that attachment

Aims:  To develop a rapid method to quantify the attachment of the cystic fibrosis pathogen, Burkholderia multivorans, to lung epithelial cells (16HBE14o−) using real-time PCR with a view to monitoring potential inhibition of lung cell attachment. Methods and Results:  Mammalian and bacterial DNA were purified from bacteria attached to lung epithelial cells. The relative amount of bacteria attached was determined by amplification of the recA gene relative to the human GAPDH gene, in the presence of SYBR Green®. The method was thoroughly validated and shown to correlate well with traditional plating techniques. Inhibition of bacterial attachment with simple sugars was then evaluated by real-time PCR. Of the sugars examined, pre-incubation of B. multivorans with lactose, mannose and xylitol all decreased bacterial adherence to 16HBE14o− cells, while glucose and galactose had no significant effect. Pre-incubation with lactose had the greatest effect, resulting in reduced adhesion to 35% of untreated controls. Conclusions:  This method can be used to quickly and effectively screen novel agents with higher affinities for bacterial adhesins. Significance and Impact of the Study:  This method will enable the rapid development of novel agents to inhibit colonization by this pathogen from the environment.