No two are alike: on the role of <i>Klebsiella pneumoniae</i> permeability barriers in antibiotic susceptibility and persistence

<i>Klebsiella pneumoniae</i> and <i>Escherichia coli</i> cause a broad range of human infections with multidrug-resistant strains presenting serious therapeutic challenges in clinics. The discovery of new antibiotics effective against these pathogens is hindered because of effective drug permeability barriers comprising active efflux pumps acting synergistically with the low-permeability outer membrane. In this study, we characterized these barriers in <i>K. pneumoniae</i> American Type Culture Collection (ATCC) 43816, a hypervirulent strain broadly used in infection models. For this purpose, we constructed an efflux-deficient strain lacking the outer membrane channel TolC, which is required for activities of most resistance-nodulation-division efflux pumps, a hyperporinated strain producing a large non-specific pore in the outer membrane, and a double-compromised strain combining these two features. We next compared the contributions of the permeability barriers of ATCC 43816 to those of the laboratory <i>E. coli</i> BW25113 in antibiotic susceptibilities and persistence and in the intracellular accumulation of fluorescent probes. We identified significant differences between these two related species. Our results show that the outer membranes of <i>K. pneumoniae</i> and <i>E. coli</i> differ in their permeability to fluorescent probes and antibiotics and that <i>K. pneumoniae</i> is more effective in efflux of various compounds. We further found that nutrient-rich and minimal growth media do not affect permeation of all antibiotics in the same way and that the mechanism of antibiotic action and specific physicochemical properties of each compound play a defining role. Likewise, the roles of permeability barriers in the persistence of <i>K. pneumoniae</i> and <i>E. coli</i> vary, depending on the mechanism of antibiotic action, its external concentration, and the affected barrier.