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.