Genetic adaptation of hypermutable pseudomonas aeruginosa colonising the airways of cystic fibrosis patients attending private and public clinics in Gauteng

Abstract

Hypermutable Pseudomonas aeruginosa (P. aeruginosa) strains harbouring defective mismatch repair (MMR) systems are prevalent in people with cystic fibrosis (pwCF). These hypermutable strains have an increased mutation rate which may enable rapid adaptation to the hostile CF lung environment. Beneficial mutations within genes conferring antibiotic resistance can occur and lead to higher antibiotic resistance and poorer clinical outcomes.

Limited data is available on hypermutable P. aeruginosa strains in African countries. The aim of this study was to determine the genetic adaptation of hypermutable P. aeruginosa colonising the airways of pwCF attending private and public clinics in South Africa using phenotypic antibiotic susceptibility methods and molecular whole genome sequencing methods to describe the mutations within the MMR genes and genes associated with antibiotic resistance as well as the genetic relatedness of the hypermutator strains.

This study consisted of 126 P. aeruginosa isolates obtained from pwCF. Hypermutable P. aeruginosa strains were screened for by determining rifampicin mutation frequencies. Antibiotic susceptibility profiles were determined using disc diffusion, broth microdilution (BMD) and the Vitek® 2 automated system (bioMérieux, France). Whole genome sequencing (WGS) was used to confirm hypermutation by analysing MMR gene mutations and genes associated with antibiotic resistance as well as determine phylogenetic relationships between hypermutable strains.

This study found a prevalence of 29% (36/126) of hypermutable isolates. It was found that 75% (27/36) of hypermutable isolates (7 private isolates and 20 public isolates) displayed multidrug resistance (MDR) or extensive drug resistance (XDR) with public isolates exhibiting greater overall antibiotic resistance. Disc diffusion and BMD provided more accurate antibiotic susceptibility testing (AST) profiles than the Vitek® 2 automated system (bioMérieux, France). Novel mutations in the MMR mutator L (mutL), mutator S (mutS) and deoxyribonucleic acid helicase II (uvrD) genes were found. Mutations were detected in the outer membrane porin (oprD) and deoxyribonucleic acid gyrase subunit A (gyrA) genes in isolates correlating with resistance to carbapenems and fluoroquinolones, respectively. A high prevalence of mucoid expression of 89% (32/36) amongst the hypermutators was observed with many isolates carrying mutations in the biofilm formation genes Sigma factor alginate U negative regulator subunit A (mucA) and acyl-homoserine lactone synthase (rhlI). Phylogenetic analysis showed clustering of the isolates according to location. Epidemic high-risk clones ST17, ST235 and ST395, which have previously been found in pwCF, were identified within this CF population and displayed MDR. This study identified two novel sequence type (ST) in the private and public setting.

Similar mutations in genes involved in antibiotic resistance were observed across all isolates indicating convergent evolution to the the CF lung. This study was the first to characterise hypermutable P. aeruginosa strains in a subset CF population in South Africa. The results emphasized the importance of identifying and monitoring these strains. Hypermutable P. aeruginosa strains harbour mutations within genes associated with antibiotic resistance that can significantly contribute to MDR which can impact treatment outcomes and potentially worsen patient health. Increased surveillance and targeted antibiotic treatment are crucial to manage hypermutable strains in clinical settings in South Africa.