Promoter deletions of Klebsiella pneumoniae carbapenemase (KPC)-encoding genes (blaKPC -2) and efflux pump (AcrAB) on ß-lactam susceptibility in KPC-producing Enterobacteriaceae.

Klebsiella pneumoniae carbapenemase (KPC)-encoding genes containing promoter-deletions (bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) have disseminated in Enterobacteriaceae. The minimal inhibitory concentrations (MICs) to ß-lactams in clinical KPC-producing Enterobacteriaceae range from susceptible to high-level resistant, resulting in diagnostic problems. To better understand the variability in ß-lactam MICs among KPC-producing Enterobacteriaceae, three isoforms of bla(KPC-2) gene were used to transform Escherichia coli W4573 and its deletion mutant of an efflux pump (AcrAB) to examine the effects on ß-lactam susceptibility. MICs to ß-lactams in E. coli W4573 and its acrAB mutant strain increased 1- to 500-fold (MIC from 0.125 to 64 µg mL(-1) of aztreonam) in the bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) transformants compared with the cloning vector alone. However, transformants of the acrAB mutant strain remained susceptible to all ß-lactams tested except for aztreonam and carbenicillin. Levels of the three promoters' length and carbapenemase activities in the transformants harboring the bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) were correlated to the levels of ß-lactam MICs in both E. coli W4573 and its mutant of an efflux pump (AcrAB). Overall, these results suggest that promoter-deletions of bla(KPC-2) gene and AcrAB may be associated with the variability in ß-lactam MICs in KPC-producing Enterobacteriaceae.

Homeostasis of glutathione is associated with polyamine-mediated ß-lactam susceptibility in Acinetobacter baumannii ATCC 19606.

Glutathione is a tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) thiol compound existing in many bacteria and maintains a proper cellular redox state, thus protecting cells against toxic substances such as reactive oxygen species. Polyamines (spermine and spermidine) are low-molecular-weight aliphatic polycations ubiquitously presenting in all living cells and modulate many cellular functions. We previously reported that exogenous polyamines significantly enhanced ß-lactam susceptibility of ß-lactam-associated multidrug-resistant Acinetobacter baumannii. In this study, three genes differentially associated with the polyamine effects on ß-lactam susceptibility were identified by transposon mutagenesis of A. baumannii ATCC 19606. All three genes encoded components of membrane transport systems. Inactivation of one of the genes encoding a putative glutathione transport ATP-binding protein increased the accumulation of intracellular glutathione (∼150 to ∼200%) and significantly decreased the polyamine effects on ß-lactam susceptibility in A. baumannii ATCC 19606. When the cells were grown with polyamines, the levels of intracellular glutathione in A. baumannii ATCC 19606 significantly decreased from ∼0.5 to ∼0.2 nmol, while the levels of extracellular glutathione were correspondingly increased. However, the levels of total glutathione (intra- plus extracellular) were unchanged when the cells were grown with or without polyamines. Overall, these results suggest that exogenous polyamines induce glutathione export, resulting in decreased levels of intracellular glutathione, which may produce an improper cellular redox state that is associated with the polyamine-mediated ß-lactam susceptibility of A. baumannii. This finding may provide a clue for development of new antimicrobial agents and/or novel strategies to treat multidrug-resistant A. baumannii.