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