2.2 Pseudomonas aeruginosa isolation and analysis
A single sputum specimen was collected from CF patients by means of spontaneous expectoration or after the administration of percussion exercises, with the assistance of the attending physiotherapist at the CF clinic. The sputum specimens were transported on ice to the Department of Medical Microbiology, University of Pretoria and cultured upon arrival on Pseudomonas CN agar (Oxoid, UK) and incubated (Vacutec, South Africa) at 37ºC for up to 72 h. Up to 10 presumptiveP. aeruginosa colonies of varying size and morphology were selected from the Pseudomonas CN agar (Oxoid, UK) plate of each CF patient sputum and sub-cultured onto 5% sheep blood agar (Diagnostic Media Products, South Africa) and incubated (Vacutec, South Africa) for up to 48 h. Gram staining was performed on the presumptive colonies to confirm purity following incubation and DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, USA).Pseudomonas aeruginosa species confirmation was performed by targeting the species-specific peptidoglycan associated outer membrane lipoprotein (oprL ) gene using primers previously described.16 The PCR reactions were prepared according to the Bioline® mastermix (Bioline®, UK) protocol and amplification of the oprL gene was performed in a Bio-Rad T100 thermocycler (Bio-Rad, USA) using the following cycling conditions: 95ºC for 5 min; 28 cycles of 95ºC for 30 s, 57ºC for 30 s, 72ºC for 1 min; and 72ºC for 10 min. The amplified PCR products were resolved on 1.5% (m/v) SeaKem® LE agarose (Lonza, USA) gel stained with 0.5 µg/µL ethidium bromide and a 100 bp molecular weight marker (ThermoScientific, USA) was used as a size reference. Pseudomonas aeruginosa ATCC 27853 was used as a positive control.
Antibiotic susceptibility testing (AST) was performed on PCR confirmedP. aeruginosa isolates using the disk diffusion technique for the following antibiotics: cefepime (30 µg), ceftazidime (30 µg), imipenem (10 µg), meropenem (10 µg), amikacin (30 µg), gentamicin (10 µg), tobramycin (10 µg), ciprofloxacin (5 µg), piperacillin/tazobactam (110 µg) and aztreonam (30 µg) and the broth microdilution technique for colistin according to the Clinical and Laboratory Standards Institute guidelines17. Multidrug resistant (MDR)P. aeruginosa isolates were defined as those displaying resistance to one or more anti-pseudomonal antibiotics in at least three or more antibiotic classes, while extensively drug resistant (XDR) isolates were those displaying resistance to one or more anti-pseudomonal antibiotics in all but two or less antibiotic classes.18,19