Impact of inducible blaDHA-1 on susceptibility of Klebsiella pneumoniae clinical isolates to LYS228 and identification of chromosomal mpl mutations mediating upregulation of plasmid borne DHA-1 expression

A panel of twenty three K. pneumoniae clinical isolates harboring plasmid-borne inducible β-lactamase DHA-1 (blaDHA-1) exhibited a wide range of susceptibilities to the novel monobactam LYS228 (MIC range 0.125 - >64 µg/mL). This panel was considerably less susceptible (MIC ≥ 8 µg/mL for 9/23 of the isolates) than was a previously reported Enterobacteriaceae strain panel comprised of 88 isolates expressing ESBLs, KPCs and MBL (MIC90 of 2 µg/mL), suggesting that blaDHA-1 can impact LYS228 susceptibility in clinical isolates. Mutants with decreased in vitro susceptibility to LYS228 and upregulated expression of blaDHA-1 were selected in vitro from K. pneumoniae blaDHA-1 strains. These had mutations in the chromosomal peptidoglycan recycling gene mpl. Pre-existing mpl mutations were identified among our clinical strains and these had strongly upregulated expression of blaDHA-1 and reduced susceptibility to LYS228. Therefore we have identified a novel mechanism of blaDHA upregulation in K. pneumoniae clinical isolates, furthering our understanding of the factors underlying β-lactam resistance and the variability in β-lactam susceptibility among these clinical strains

Target (MexB) and efflux based mechanisms decreasing the effectiveness of the efflux pump inhibitor D13-9001 in P. aeruginosa PAO1: uncovering a new role for MexMN-OprM in efflux of β-lactams and a novel regulatory circuit controlling MexMN expression

Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13- 9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a �150-fold upregulation of MexMN pump gene expression and a �50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several �-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance

Design, Synthesis and Properties of a Potent Inhibitor of Pseudomonas aeruginosa Deacetylase LpxC

Over the past several decades the frequency of bacterial antimicrobial resistance in hospitals, including resistance to multiple antibacterials (multi-drug resistant, MDR) and its association with serious infectious diseases, have increased at alarming rates. Pseudomonas aeruginosa is a leading cause of nosocomial infections, and resistance to virtually all approved antibacterials is emerging in this pathogen. To address the need for new agents to treat MDR P. aeruginosa, we focused on inhibiting the first committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by the enzyme LpxC. We approached this through the design, synthesis and biological evaluation of several potent hydroxamic acid LpxC inhibitors, such as 1

Design, Synthesis, and Properties of a Potent Inhibitor of <i>Pseudomonas aeruginosa</i> Deacetylase LpxC

Over the past several decades, the frequency of antibacterial resistance in hospitals, including multidrug resistance (MDR) and its association with serious infectious diseases, has increased at alarming rates. <i>Pseudomonas aeruginosa</i> is a leading cause of nosocomial infections, and resistance to virtually all approved antibacterial agents is emerging in this pathogen. To address the need for new agents to treat MDR <i>P. aeruginosa</i>, we focused on inhibiting the first committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphospho-3-<i>O</i>-(<i>R</i>-hydroxydecanoyl)-<i>N</i>-acetylglucosamine by the enzyme LpxC. We approached this through the design, synthesis, and biological evaluation of novel hydroxamic acid LpxC inhibitors, exemplified by <b>1</b>, where cytotoxicity against mammalian cell lines was reduced, solubility and plasma-protein binding were improved while retaining potent anti-pseudomonal activity in vitro and in vivo