Klebsiella pneumoniae is a deadly, nosocomial pathogen causing severe respiratory infections and bacteraemia. The WHO has flagged multidrug-resistant (MDR) K. pneumoniae strains as an "urgent threat to human health". Resistance to all classes of antimicrobials is on the rise, meaning that infections are becoming increasingly difficult to treat. Thus, combination therapy is becoming an attractive last-line option.
Here, we use Transposon Directed Insertion Sequencing (TraDIS) to assay the contribution of each chromosomal gene in a MDR clinical isolate of the K. pneumoniae epidemic clone ST258 to its survival during antibiotic exposure. A dense TraDIS library of over 300,000 unique Tn5 mutants was exposed to two passages in Mueller-Hinton broth (MHB) containing ciprofloxacin (cip), amikacin (amk) or imipenem (imp), either alone or in combination (cip+amk or cip+imp). Each drug was added to a medium at a sub-inhibitory concentration of antibiotic, and MHB without antimicrobial was used as a control. Sequencing was performed on a HiSeq2500 Illumina platform, and reads were mapped back onto the PacBio-sequenced reference genome, comparing insertion numbers for each gene in the treated cultures and the untreated control.
We examined genes that are only important for survival during exposure to antimicrobial combinations. A number of genes displayed a significant reduction in insertion mutants, only when the strain was exposed to both antimicrobial combinations, indicating “synergy-specific resistance”. We confirmed, using single gene knock outs, that one representative gene (ihfB) was involved in resistance only during combinational treatment. We also identified genes involved in sensitivity to these combinations.
This study pushes forward the emerging field of antibiotic combination therapy by providing insights into the survival strategies of MDR K. pneumoniae during exposure to specific antimicrobial combinations. The information generated by the study opens future avenues to finding novel antimicrobial helper drug targets able to enhance the activity of known antimicrobial combinations against this important nosocomial pathogen.