Polymyxins, including polymyxin B and polymyxin E (colistin), have been pivotal in the treatment of multidrug-resistant Gram-negative bacteria(1). Resistance towards these antibiotics is rapidly emerging, particularly in extensively drug-resistant Klebsiella pneumoniae, resulting in high mortality(2). Utilizing whole genome sequencing, we interrogated the genomes of 24 clinical isolates from Greece and Brazil. Nineteen isolates harboured polymyxin resistance which was enabled by mutations in the two-component regulatory systems pmrAB, phoPQ and the negative regulator, mgrB. In approximately 80% of these isolates, mgrB was perturbed either by missense mutations, nonsense mutations or insertion sequences. Several classical and novel variants were detected. Interestingly, up to 3 predicted deleterious alterations were identified in differing genes within individual isolates. Additional mutations did not correlate to an increase in minimal inhibition concentration (MIC) towards polymyxins.
We further selected a polymyxin susceptible strain (MIC: 0.125mg/ L) and passaged in the presence of either polymyxin B or colistin for 20 days. Different trends for resistance acquisition were observed, although all eventually harboured a polymyxin resistant phenotype (MIC: >2mg/ L) with 5 of 6 replicates from each group possessing a substantial MIC of >128mg/ L. Four colonies from 4 day 20 replicate treatment groups were selected for sequencing. Deleterious missense mutations were apparent in pmrB, phoP, phoQ and mgrB. Notably, 3 replicates had dual hits in 2 different genes, one replicate had a complete deletion of the mgrB locus and one replicate from each treatment group had no aberrations in these genes. This was evident in all 4 colonies from each replicate indicating selective clonal expansion. None of these alterations occurred in the same position as reported from clinical isolates.
Clinical and induced strains appear to be commonly altered in pmrB, phoP, phoQ and mgrB in various positions. Predicted deleterious hits can arise in a combination of genes, however multiple changes did not directly correlate with MIC. Polymyxin resistance separate to these genes was detected inferring potential novel pathways involved.