In vitroevolution of colistin resistance in theKlebsiella pneumoniaecomplex follows multiple evolutionary trajectories with variable effects on fitness and virulence characteristics
Authors: Janssen Axel B. , Doorduijn Dennis J. , Mills Grant , Rogers Malbert R.C. , Bonten Marc J.M. , Rooijakkers Suzan H.M. , Willems Rob J.L. , Bengoechea Jose A. , Schaik Willem van 
Published: May 2020

The increasing prevalence of multidrug-resistant Gram-negative opportunistic pathogens, includingKlebsiella pneumoniae, has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic lipopeptide antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS). Colistin resistance inK. pneumoniaeis mediated through loss of these phosphate groups, or modification with cationic groups (e.g. 4-amino-4-deoxy-L-arabinose (L-Ara4N), or phosphoethanolamine), but also hydroxylation of acyl-groups of lipid A. Here, we study thein vitroevolutionary trajectories towards colistin resistance in clinicalK. pneumoniaecomplex strains (threeK. pneumoniae sensu strictostrains and oneK. variicolasubsp.variicolastrain) and their impact on fitness and virulence characteristics.

Through population sequencing during thein vitroevolution experiment, we found that resistance develops through a combination of single nucleotide polymorphisms (SNPs), insertion and deletions (indels), and the integration of insertion sequence (IS) elements, affecting genes associated with LPS biosynthesis and modification, and capsule structures. The development of colistin resistance decreased the maximum growth rate of oneK. pneumoniae sensu strictostrain, but not in the other threeK. pneumoniae sensu latostrains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and L-Ara4N addition. Colistin-resistantK. pneumoniae sensu strictostrains exhibited cross-resistance to LL-37, in contrast to theK. variicolasubsp.variicolastrain that did not change in susceptibility to LL-37. Virulence, as determined in aCaenorhabditis eleganssurvival assay, was higher in two colistin-resistant strains.

Our study suggests that nosocomialK. pneumoniaecomplex strains can rapidly develop colistin resistancede novothrough diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the lifespan of this last-resort antibiotic for the treatment of infections with multidrug-resistantKlebsiella.

Author summary

Bacteria that frequently cause infections in hospitalised patients are becoming increasingly resistant to antibiotics. Colistin is a positively charged antibiotic that is used for the treatment of infections with multidrug-resistant Gram-negative bacteria. Colistin acts by specifically interacting with the negatively charged LPS molecule in the outer membrane of Gram-negative bacteria. Colistin resistance is mostly mediated through modification of LPS to reduce its negative charge. Here, we use a laboratory evolution experiment to show that strains belonging to theKlebsiella pneumoniaecomplex, a common cause of multidrug-resistant hospital-acquired infections, can rapidly accumulate mutations that reduce the negative charge of LPS without an appreciable loss of fitness. Colistin resistance can lead to cross-resistance to an antimicrobial peptide of the human innate immune system, but can increase susceptibility to serum, and virulence in a nematode model. These findings show that extensively resistantK. pneumoniaecomplex strains may rapidly develop resistance to the last-resort antibiotic colistin via different evolutionary trajectories, while retaining their ability to cause infections.