Pandoraea species are emerging pathogens in cystic fibrosis (CF) patients, however, the contribution to disease progression remains elusive1. Current treatments comprise of imipenem and trimethoprim-sulfamethoxazole (co-trimoxazole). A new species has recently been identified, Pandoraea fibrosis, but the conventional treatment regime was unsuccessful clinically2. This study aimed to assemble the genome, investigate the transcriptome, expand the panel of antimicrobial susceptibility testing and evaluate the progression of resistance.
The sequential P. fibrosis clinical isolates 6399 and 7641 were obtained from a CF patient (Royal Hobart Hospital, Tasmania, Australia)3. DNA was extracted using the DNeasy Blood and Tissue kit, RNA via the RNeasy Mini Kit with an additional rRNA depletion and poly(A) tailing for direct RNA sequencing. Samples were sequenced on a MinION (Oxford Nanopore Technologies) sequencer. Combined with prior Illumina sequencing4, genomes were assembled using Unicycler, variants determined via GATK (impact using snpEff) and resistance genes detected with ResFinder3.1. As no breakpoints (CLSI, EUCAST guidelines) exist for Pandoraea, resistance was determined using closely related species. Evaluating the progression of resistance was performed by passaging isolates in increasing concentrations of either imipenem or co-trimoxazole for 10 days followed by 3 antibiotic-free passages (n=4).
The completed genome assembly for each isolate was a singular 5.59 Mb circular contig. Genome length differed by one nucleotide and three missense mutations. ResFinder detected one gene conferring resistance to beta-lactams, blaOXA-154 (82% identity). The top differentially expressed genes were associated with efflux pumps (≥1.4-fold increase in 7641). Of the 40 antibiotics assayed, in vitro susceptibility was identified for imipenem, co-trimoxazole, doxycycline and minocycline. Selection of resistance revealed one 7641 replicate developing resistance against imipenem at day 7 and MICs were similar to MBCs. Co-trimoxazole non-susceptibility was observed temporarily at day 9 for one 7641 replicate, however, was unstable and MBCs were commonly ≥4-fold higher than MICs.
Overall, this study has provided further insight into the genome, resistome and transcriptome of P. fibrosis.