Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant<i>Klebsiella pneumoniae</i>and<i>Enterobacter cloacae</i>

Abstract

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against <i>Klebsiella pneumoniae</i> and <i>Enterobacter cloacae</i>, there are limited data on OM permeability especially in <i>K. pneumoniae</i> Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant <i>K. pneumoniae</i> and <i>E. cloacae</i> Both clinical isolates harbored a <i>bla</i>_KPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our <i>K. pneumoniae</i> isolate was polymyxin resistant, whereas the <i>E. cloacae</i> was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated <i>E. cloacae</i> at least 258-fold faster and <i>K. pneumoniae</i> 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the <i>E. cloacae</i> compared to the <i>K. pneumoniae</i> isolate (except for aztreonam). This correlated with a higher OmpC porin production in <i>E. cloacae</i>, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant <i>K. pneumoniae</i> and <i>E. cloacae</i> Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics.<b>IMPORTANCE</b> Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of <i>Klebsiella pneumoniae</i> and <i>Enterobacter cloacae</i>, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of <i>K. pneumoniae</i> and <i>E. cloacae</i> substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.