The secondary resistome of multidrug-resistant Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the “secondary resistome”. As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial “helper” drugs that restore the efficacy of existing antimicrobials.

A putative RND-type efflux pump, H239_3064, contributes to colistin resistance through CrrB in Klebsiella pneumoniae

“… 16 The additional loci identified in the present study included dedA , which encodes a putative integral membrane protein; the previous study had demonstrated that dedA is essential for growth during exposure to colistin. 32 usg was also identified by a transposon insertion in the present study; notably, usg is located upstream of dedA in the K. pneumoniae genome, so insertion at usg may have polar effects on dedA expression. Multiple additional loci associated with colistin resistance were also identified for the first time in the present study.…”

Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis

“…To answer this question, we performed transposon (Tn)-directed insertion sequencing (TraDIS) using the clinical bloodstream isolate ID40, which is resistant to many ␤-lactam antibiotics, to assess the resistome of P. aeruginosa by an approach similar to that described by Jana et al (17). TraDIS has been shown to be a valuable tool under particular conditions and in various approaches to find genes responsible for growth (18)(19)(20)(21).…”

Repurposing azithromycin and rifampicin against Gram-negative pathogens by combination with peptide potentiators

“…ATCC reference strains included Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. MDR strains E. coli ST131 and K. pneumoniae ST258 are clinical isolates from urinary tract [7] and wound infections [8], respectively. A panel of β-lactamase resistant clinical isolates of were provided by Laurent Poirel.…”