4 DISCUSSION
The CF clinic based at the study setting practices patient segregation
based on the segregation of patients colonised withP. aeruginosa, from those not colonised with P.
aeruginosa. This is achieved by scheduling clinics for each of the
patient groups on different weeks. However, due to a lack of frequent
genomic surveillance of P. aeruginosa CF isolates in South
Africa, limited knowledge is available on the presence of epidemic
strains being carried by CF patients.
The CF patients from which the P. aeruginosa AUST-03 strains had
been detected were children, who were both experiencing pulmonary
exacerbations. Limited data is available on the patient demographics of
CF patients infected or colonised with P. aeruginosa AUST-03;
however, the studies that are available have reported that P.
aeruginosa AUST-03 has been associated with increased pulmonary
exacerbations.12,30 Only a single study was found in
the literature that investigated P. aeruginosa AUST-03 (then
referred to as AES-III) by Bradbury and colleagues30,
however, clear comparisons in the age demographics of the CF patients
from that study and the current study could not be made. This was due to
Bradbury et al.30 having only recruited patients
during outreach clinics for adult CF patients, while the CF clinics from
this study only attended to children with CF. Bradbury et
al.30 did recruit patients as young as 15 years of age
in the study, however, those that were found to harbourP. aeruginosa AUST-03 strains were between the ages of 19 and 34
years old. Furthermore, only a very small number of patients carrying
this strain were detected in the current study.
P2 was found to be exclusively colonised with P. aeruginosaAUST-03, while P4 was found to be colonised with P. aeruginosaAUST-03 and a second novel strain. Bradbury et al.30also made a similar observation in one of their patients and found that
the patient was infected with three different P. aeruginosastrains, including AUST-03. Phenotypic heterogeneity was observed among
the ten P. aeruginosa AUST-03 isolated from P2 with an observed
mixture of mucoid (7/10) and non-mucoid (3/10) isolates of varying size.
Variations in the presence of mucoidity among P. aeruginosaAUST-03 strains have also been reported in other
studies15,30 and similarly to our study mucoid
isolates were no more resistant to antibiotics that non-mucoid isolates.
Multiple studies have reported an increased likelihood of P.
aeruginosa AUST-03 strains displaying a MDR phenotype through their
antibiograms.12,15,30 A similar observation was found
in this study where the majority (8/11) of the P. aeruginosaAUST-03 isolates were MDR or XDR. The study isolates were most
frequently resistant to ciprofloxacin [100% (11/11)], cefepime
[73% (8/11)], gentamicin [64% (7/11)] and infrequently
resistant to tobramycin [18% (2/11)]. Similar findings were
reported by Bradbury et al.30; however, unlike in the
Bradbury et al.30 study, low rates of resistance were
observed to amikacin [36% (4/11)], ceftazidime [18% (2/11)],
imipenem [18% (2/11)] and aztreonam [9% (1/11)]. This
variation in the two studies may be due to differences in treatment
regimens between Australia and South Africa, as well as the differences
in time periods (2019 for this study and 2003 for the Bradbury et
al.30 study), which may have affected the availability
of certain antibiotics. The high rates of ciprofloxacin resistance may
be due to the frequent use of ciprofloxacin in South African settings as
this antibiotic is recommended for the treatment of P. aeruginosaexacerbations.6 Inhaled gentamycin is recommended by
the South African Cystic Fibrosis Consensus
Guidelines6 for the eradication and chronic
suppressive treatment of P. aeruginosa in CF patients due to the
limited availability of inhaled tobramycin. This may explain the high
rate of resistance to gentamycin. Additionally, the limited
accessibility of tobramycin at public CF clinics in South Africa may
also explain the low rates of tobramycin resistance among the P.
aeruginosa AUST-03 strains.
Multidrug efflux pumps formed the basis of antibiotic resistance in the
genomes of the P. aeruginosa AUST-03 strains from this study. The
most clinically relevant efflux pumps were: MexAB-OprM (extrudes
aztreonam, ceftazidime, ciprofloxacin, levofloxacin, meropenem and
piperacillin), MexCD-OprJ (extrudes azithromycin, cefepime,
ciprofloxacin, levofloxacin), MexEF-OprN (extrudes ciprofloxacin and
imipenem) and MexXY-OprM (extrudes amikacin, cefepime, ciprofloxacin,
gentamicin, levofloxacin and tobramycin), which were detected in the 11
isolates.31,32 Furthermore, mutations in the
regulatory genes: nalC (S209R and G71E), nfxB (Type A),mexRT of the 11 study isolates were detected that conferred the
overexpression of the MexAB-OprM, MexCD-OprJ and MexEF-OprN efflux
pumps, respectively.31,32 A limited number of
clinically relevant acquired ARGs were detected in the study isolates,
namely crpP (11/11) which confers resistance to ciprofloxacin andPDC-3 (11/11) which confers resistance to
piperacillin/tazobactam.33-35 Acquired mutations in
the DNA gyrase [gyrA_D87N (3/11) and gyrB (8/11)] and
topoisomerase IV [parE (1/11)] genes were detected in theP. aeruginosa AUST-03 strains, conferring resistance to
fluoroquinolones such as ciprofloxacin.36
Colistin is considered to be an antibiotic of last resort for the
treatment of Gram-negative pathogens such as P. aeruginosa in
healthcare
settings.37 As
such, the use of this antibiotic is reserved for MDR isolates, where all
other treatment options are ineffective. In this study, only a singleP. aeruginosa AUST-03 strain (P4) was resistant to colistin and
was also found to be XDR, regrettably, this patient passed away. In this
patient’s strain, mutations were found in the lipid A regulatory genes
(pmrAB ) that conferred enhanced colistin
resistance.36 Mutations in these genes were also found
in the other 10 P. aeruginosa AUST-03 strains (P2) from this
study, however, these isolates displayed intermediate resistance to
colistin. The presence of these mutations could lead to the future
development of colistin resistance in the strains.
The three strains from P2 and the strain from P4 clustered together in
Cluster 2, which may indicate that these were the initial strains that
were acquired by the two patients. This may have been due to strain
transfer among the two patients or each patient acquiring this strain at
the same time from the same external source. The countries Canada,
China, Denmark and Slovenia were also part of this cluster and could
have potentially been involved into the introduction of this strain to
the study setting, however, further investigation is required. Strains
from Canada were a common feature in the three clusters (Clusters 2, 3
and 7) containing strains from this study and global strains. Canada is
a popular destination for migration from many African countries
including South Africa38 and the frequent back and
forth travel between the two countries may present opportunities for the
introduction of P. aeruginosa AUST-03 in CF clinics. However,
introduction from other countries or multiple countries cannot be ruled
out. None of the P. aeruginosa ST242 (AUST-03) strains from this
study clustered with the Australian strains at a cut-off of 20 SNP
differences, however, the strains were still closely related and
differences may have been due to genomic changes that occurred over time
and over the spread of the strain globally. Infection control strategies
in South Africa will need to be revised to include the screening of
patients that may have travelled or lived in countries where epidemic
strains are endemic.
The main limitation from the study was the low numbers of CF patients
that were investigated and that adult CF patients from this hospital
were not included in the study as P. aeruginosa AUST-03 was found
mostly in adult CF patients in previous studies.30Furthermore, the limited attention given to strain typing of P.
aeruginosa in the study setting makes it difficult to ascertain when
the P. aeruginosa AUST-03 first appeared in the study setting and
for how long this epidemic strain has been circulating. Future studies
investigating P. aeruginosa AUST-03 in minor and adult CF
patients at other public and private CF clinics across the country are
important to establish the spread of this and other epidemic strains.