Trade-offs between antibacterial resistance and fitness cost in the production of metallo-b-lactamases by enteric bacteria manifest as sporadic emergence of carbapenem resistance in a clinical setting

Meropenem is a clinically important antibacterial reserved for treatment of multiresistant infections. In meropenem-resistant bacteria of the family Enterobacterales, NDM-1 is considerably more common than IMP-1, despite both metallo-β-lactamases (MBLs) hydrolyzing meropenem with almost identical kinetics. We show that bla(NDM-1) consistently confers meropenem resistance in wild-type Enterobacterales, but bla(IMP-1) does not. The reason is higher bla(NDM-1) expression because of its stronger promoter. However, the cost of meropenem resistance is reduced fitness of bla(NDM-1)-positive Enterobacterales. In parallel, from a clinical case, we identified multiple Enterobacter spp. isolates carrying a plasmid-encoded bla(NDM-1) having a modified promoter region. This modification lowered MBL production to a level associated with zero fitness cost, but, consequently, the isolates were not meropenem resistant. However, we identified a Klebsiella pneumoniae isolate from this same clinical case carrying the same bla(NDM-1) plasmid. This isolate was meropenem resistant despite low-level NDM-1 production because of a ramR mutation reducing envelope permeability. Overall, therefore, we show how the resistance/fitness trade-off for MBL carriage can be resolved. The result is sporadic emergence of meropenem resistance in a clinical setting

Comparative effects of overproducing the AraC-type transcriptional regulators MarA, SoxS, RarA and RamA on antimicrobial drug susceptibility in Klebsiella pneumoniae

OBJECTIVES: In Klebsiella pneumoniae, overproduction of RamA and RarA leads to increased MICs of various antibiotics; MarA and SoxS are predicted to perform a similar function. We have compared the relative effects of overproducing these four AraC-type regulators on envelope permeability (a combination of outer membrane permeability and efflux), efflux pump and porin production, and antibiotic susceptibility in K. pneumoniae. METHODS: Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT–PCR. RESULTS: Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific. CONCLUSIONS: RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates