Impact of meropenem on Klebsiella pneumoniae metabolism

Abstract

The aim of this study was to analyze the metabolome of several Klebsiella pneumoniae strains characterized by different resistance patterns. A total of 59 bacterial strains (27 carbapenemase-negative and 32 carbapenemase-positive) were included and their metabolic features were assessed in basal conditions. Moreover, 8 isolates (4 wild-type and 4 KPC-producers) were randomly selected to evaluate the impact of sub-lethal concentrations of meropenem on bacterial metabolism. The metabolomic analysis was performed by 1H-NMR spectroscopy both on filtered supernatants and cell lysates. A total of 40 and 20 molecules were quantified in the intracellular and the extracellular metabolome, respectively. While in basal conditions only five metabolites showed significant differences between carbapenemase-positive and negative strains, the use of meropenem had a profound impact on the whole bacterial metabolism. In the intracellular compartment, a reduction of different overflow metabolites and organic acids (e.g. formate, acetate, isobutyrate) was noticed, whereas, in the extracellular metabolome, the levels of several organic acids (e.g. succinate, acetate, formate, lactate) and amino acids (aspartate, threonine, lysine, alanine) were modified by meropenem stimulation. Interestingly, carbapenemase-positive and negative strains reacted differently to meropenem in terms of number and type of perturbed metabolites. In wild-type strains, meropenem had great impact on the metabolic pathways related to methane metabolism and alanine, aspartate and glutamate metabolism, whereas in KPC-producers the effect was predominant on pyruvate metabolism. The knowledge about the bacterial metabolic profiles could help to set up innovative diagnostic methods and new antimicrobial strategies to fight the global crisis against carbapenemase-positive K. pneumoniae.

Fig 1

rPCA model calculated on the space constituted by the concentration of the intracellular (upper panel) and extracellular (lower panel) molecules that significantly differed between non-stressed and meropenem-stressed bacterial strains. In the scoreplot (left), non-stressed strains and meropenem-stressed strains are represented with black square and red dots respectively, with lines connecting each strain to the median of its group. In the barplot (right), describing the correlation between the concentration of each molecule and its importance over PC1, dark gray bars highlight statistically significant correlations (P<0.05). In the barplot, bars pointing to the left denote metabolites that were more abundant in non-stressed cells, while bars pointing to the right denote molecules that were more abundant in meropenem-stressed bacteria.

Fig 2. Metabolome view of the metabolic pathways altered after meropenem exposure in the group of the 8 selected K. pneumoniae strains.

Each dot represents a unique metabolic pathway, with the dot size corresponding to the pathway impact score and the dot color (red being the most significant) corresponding to the–log(P) value.

Fig 3

Metabolome view of the metabolic pathways altered after meropenem exposure in the group of wild-type (left) and KPC-producing (right) K. pneumoniae strains. Each dot represents a unique metabolic pathway, with the dot size corresponding to the pathway impact score and the dot color (red being the most significant) corresponding to the–log(P) value.