Programmpunkt

Titel: Intraclonal competitive fitness of Pseudomonas aeruginosa cystic fibrosis airway isolates in human precision-cut lung slices (PCLS)
ID: 183/MPP
Art: Abstractautor
Session: P1
Microbial Pathogenesis (FG MP)

Referent: Marie Luise Nawrot (Hannover)


Abstract - Text

Chronic airway infections with the gram-negative bacterium Pseudomonas aeruginosa (P. aeruginosa) play an important role for the clinical course of patients suffering from cystic fibrosis (CF). In this case, infections of the respiratory tract cause progressive loss of lung function and reduced life expectancy. During the colonization of the patient"s lung, the bacteria undergo microevolution, presumably to adapt to the CF lung habitat. After analyzing the influence of this adaptation on the fitness of P. aeruginosa to grow together in an aquatic habitat (in vitro), we now want to explore how the competitive fitness of the bacteria looks like, if they are grown in a human lung habitat.

For this purpose, the PCLS will be used as a human based ex vivo model. The PCLS will be prepared from human explanted lung tissue, filled with agarose gel and cut into small slices. Several experiments have proven that these slices are still viable and show functional responses, like airway- and vasoconstriction or immunological and epithelial reactions*. After pretreatment with media, the slices will be infected with competitively growing serial clonal P. aeruginosa isolates from 12 patients (6 with severe courses and 6 with mild courses). These isolates were selected from P. aeruginosa positive CF-patients in the clinic at the Hannover Medical School during their whole infection period – exemplary up to 30 years. To evaluate the composition of the bacterial communities after 12 and 24 hours, the slices will be homogenized, dilutions will be made and plated on LB-agar plates. Afterwards, the bacterial DNA will be isolated and the relative quantities of each sequential isolate will be determined by sequencing of bacterial DNA-derived amplicons spanning strain-specific SNPs.

 

* Neuhaus, V., Danov, O., Konzok, S., Obernolte, H., Dehmel, S., Braubach, P., Jonigk, D., Fieguth, H.G., Zardo, P., Warnecke, G., Martin, C., Braun, A., Sewald, K. Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices. J. Vis. Exp. (), e57042, doi:10.3791/57042 (2018)