Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile

Abstract

Background and Objectives: Respiratory infections are the most serious condition in cystic fibrosis (CF) patients; therefore, a thorough comprehension of the diversity and dominant microbial species in CF airways has a crucial role in treatment. Our objective was to determine the antibiotic resistance profile of CF airways microbiota and compare culture methods and PCR-DGGE to evaluate bacterial diversity. Materials and Methods: Pharyngeal swabs from 121 CF patients were collected. The samples were then cultured, identified and antibiotic resistance testing was performed. Thirty samples were subjected to further molecular surveys. DNA contents of these samples were extracted and amplified using nested-PCR technique and their bacterial diversity was assessed by DGGE. The DGGE patterns were visualized and certain bands were excised and purified. Next, the DNA was amplified by another round of PCR and sent out for sequencing. Results: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the most prevalent species isolated using culture methods. S. aureus was the most common bacteria among 6 years and younger patients; while, P. aeruginosa had more prevalence among older ones. The PCR-DGGE results showed more diversity than culture methods, particularly in younger patients who exhibited more bacterial diversity than the older groups. Sequencing results unveiled the presence of certain bacterial species including Haemophilus parainfluenzae and Stenotrophomonas maltophilia which were completely missed in culture. Conclusion: Even though culture-dependent methods are cost-effective, PCR-DGGE appeared to be more efficient to determine bacterial diversity. PCR-DGGE detects less abundant species, though their viability could not be determined using this method.

Keywords

• Cystic fibrosis;
• Respiratory tract infections;
• Antibiotic resistance;
• Denaturing gradient gel electrophoresis;
• Microbiota