Evaluation of Bacterial Diversity in Cystic Fibrosis Respiratory Samples via PCR-DGGE and its Comparison with Culture Results

Background and Aim: Cystic fibrosis is the most common lethal genetic disorder, which is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR) protein-encoding gene. This protein is an ion channel that conducts chloride across the epithelial cells membrane. In the lungs, disruption of chloride transport leads to the dehydration of mucosal surface layer, which impairs mucociliary clearance. Such condition provides a proper opportunity for the colonization of bacteria and the emergence of the chronic infections. In the current study, the diversity and structure of the respiratory microbiota in CF patients were investigated by PCR-DGGE and culture-dependent techniques.Methods: ۳۵ CF patients were collected with deep pharyngeal swabs. Identification of the isolated bacteria with the culture-dependent methods was carried out in the clinical laboratory. DNA extraction was carried out using chloroform-isoamyl alcohol method. Subsequently, bacterial DNA was amplified by nested-PCR with ۲۷f/۱۴۹۲r and ۳۴۱f-GC/۷۸۲r primers respectively. The PCR products was loaded in ۸% acrylamide gel with ۳۵% to ۷۵% of the gradient concentration of urea and formamide. Electrophoresis was performed at ۶۰°C for ۷۵۰ min with ۶۰V. In each DGGE gel, the amplified DNA of the isolated S. aureus, P. aeruginosa, K. pneumoniae and, Enterobacter sp. were loaded alongside the samples as a control.Results: Comparison between the children and adult CF profiles showed a decrease in bacterial diversity with age. Culture-dependent and PCR-DGGE techniques were used to detect some bacterial groups. The number of Pseudomonas sp. identified by PCR-DGGE was significantly higher than its number identified via culturing method.Conclusion: Pseudomonas sp. was detected in more patients by the PCR-DGGE than culture-dependent method. This difference may be due to the detection of DNA from dead bacteria that are trapped in thick respiratory secretions. Besides, in response to antibiotic treatments, auxotrophic strains emerge that are not easily cultivable but can be identified by molecular methods. A decrease in bacterial diversity with age can be attributed to the continuous use of broad-spectrum antibiotics, causing selective pressure in these communities.

Keywords:

Cystic Fibrosis , Microbiota , PCR-DGGE , Molecular Identification , Respiratory Tract