A phylogenomic approach for the analysis of colistin resistance-associated genes in Klebsiella pneumoniae, its mutational diversity and implications for phenotypic resistance

The emergence of carbapenemase-producing Klebsiella pneumoniae strains has triggered the use of old antibiotics such as colistin. This is driving the emergence of colistin resistance in multidrug-resistant strains that underlie life-threatening infections. This study analyses the mutational diversity of 22 genes associated with colistin resistance in 140 K. pneumoniae clinical isolates integrated in a high-resolution phylogenetic scenario. Colistin susceptibility was accessed by broth microdilution. A total of 98 isolates were susceptible and 16 were resistant, 10 of which were carbapenemase producers. Across the 22 genes examined, 171 non-synonymous mutations and 9 mutations associated with promoter regions were found. Eighty-five isolates had a truncation and/or deletion in at least one of the 22 genes. However, only seven mutations, the complete deletion of mgrB or insertion sequence (IS)-mediated disruption, were exclusively observed in resistant isolates. Four of these (mgrB Ile13fs, pmrB Gly207Asp, phoQ His339Asp and ramA Ile28Met) comprised novel mutations that are potentially involved in colistin resistance. One strain bore a ISEcp1-blaCTX-M-15::mgrB disruption, underlying co-resistance to third-generation cephalosporins and colistin. Moreover, the high-resolution phylogenetic context shows that most of the mutational diversity spans multiple phylogenetic clades, and most of the mutations previously associated with colistin resistance are clade-associated and present in susceptible isolates, showing no correlation with colistin resistance. In conclusion, the present study provides relevant data on the genetic background of genes involved with colistin resistance deeply rooted across monophyletic groups and provides a better understanding of the genes and mutations involved in colistin resistance.This study was supported in part by UID/DTP/04138/2019 from Fundação para a Ciência e Tecnologia (FCT), Portugal. RE is supported by the FCT trough the PhD Fellowships (Grant Reference: 2021.08701.BD). JP [CEECIND/00394/2017] is supported by Fundação para a Ciência e Tecnologia through Estímulo Individual ao Emprego Científico. TGC received funding from the MRC UK (Grant no. MR/K000551/1, MR/M01360X/1, MR/N010469/1, MR/R020973/1, MR/R025576/1, MR/S01988X/1, MR/S03563X/1) and BBSRC UK (BB/R013063/1). SC received funding from the Bloomsbury Set, Medical Research Council UK grants (MR/R020973/1, MR/R025576/1, MR/S01988X/1, MR/S03563X/1) and the BBSRC UK (BB/R013063/1).