Representative 1,2′-DNA efflux curves of 3-AG100-derived AcrB binding pocket mutants.

<p>After preloading with 4 µM 1,2′-DNA the cells were energized at 100 s with 50 mM glucose. Fluorescence intensity is given as relative fluorescence units (RFU) with preenergization levels adjusted to 100 RFU.</p

Increased quenching of 1,2′-DNA fluorescence at higher dye loading concentrations (DLCs) in strain 3-AG100.

<p>Depicted are relative fluorescence units (RFU) of deenergized cells preloaded with 1,2′-DNA after resuspension in fresh PPB. All experiments were done in triplicate. The error bars show the standard deviation.</p

Results of susceptibility testing (MICs), the molecular analyses of β-lactamases and porins, the efflux properties (EHT), and the synergism testing (FICIs).

<p>The EHT and the FICI values are given as median ± standard deviation. Synergistic FICI values are indicated in bold letters. The FICI values indicated in brackets represent the lowest values for the given combination.</p><p>*Strains were obtained from: NRZ = National Reference Laboratory for multidrug-resistant gram-negative bacteria, Ruhr-University Bochum, Germany, and RKI = Robert-Koch-Institut, Wernigerode, Germany; MEM = meropenem; CST = colistin; TCG = tigecycline; FICI = fractional inhibitory concentration index; EHT = efflux half-time; nm = not measureable; aa = amino acid;:: = gene dispuption by insertion sequence; del = deletion; ins insertion; sub = substitution;</p><p>**non-functional (STOP mutation in <i>bla</i>_OXA-9 gene)</p><p>Results of susceptibility testing (MICs), the molecular analyses of β-lactamases and porins, the efflux properties (EHT), and the synergism testing (FICIs).</p

Three Dimensional Checkerboard Synergy Analysis of Colistin, Meropenem, Tigecycline against Multidrug-Resistant Clinical <i>Klebsiella pneumonia</i> Isolates

<div><p>The spread of carbapenem-non-susceptible <i>Klebsiella pneumoniae</i> strains bearing different resistance determinants is a rising problem worldwide. Especially infections with KPC (<i>Klebsiella pneumoniae</i> carbapenemase) - producers are associated with high mortality rates due to limited treatment options. Recent clinical studies of KPC-blood stream infections revealed that colistin-based combination therapy with a carbapenem and/or tigecycline was associated with significantly decreased mortality rates when compared to colistin monotherapy. However, it remains unclear if these observations can be transferred to <i>K</i>. <i>pneumoniae</i> harboring other mechanisms of carbapenem resistance. A three-dimensional synergy analysis was performed to evaluate the benefits of a triple combination with meropenem, tigecycline and colistin against 20 <i>K</i>. <i>pneumoniae</i> isolates harboring different β-lactamases. To examine the mechanism behind the clinically observed synergistic effect, efflux properties and outer membrane porin (Omp) genes (<i>ompK35</i> and <i>ompK36</i>) were also analyzed. Synergism was found for colistin-based double combinations for strains exhibiting high minimal inhibition concentrations against all of the three antibiotics. Adding a third antibiotic did not result in further increased synergistic effect in these strains. Antagonism did not occur. These results support the idea that colistin-based double combinations might be sufficient and the most effective combination partner for colistin should be chosen according to its MIC.</p></div

Exemplary isoboles of the double combinations of the antibiotics.

<p>A-C: Isoboles of strain NRZ 00246 (OXA-48 producer) exhibiting synergistic effects. D-F: Isoboles of strain RKI 318/11 (KPC producer) yielding no synergism. FIC = fractional inhibitory concentration.</p

Inhibition zones of the antibiotics in disc diffusion test of selected strains.

<p>Inhibition zones of the antibiotics in disc diffusion test of selected strains.</p

Exemplary plots of the checkerboard assays for the double combinations of the antibiotics.

<p>A-C: Strain NRZ 00246 (OXA-48 producer) exhibiting FICI <0.8. D-F: Strain RKI 318/11 (KPC producer) exhibiting FICI >0.8. Grey lines indicate the breakpoints of the respective antibiotic (according to EUCAST).</p

Sequence alignment of <i>ompK36</i> genes.

<p>The sequence of <i>ompK36</i> of <i>K</i>. <i>pneumoniae</i> strain JM45 (accession number CP006656) was used as reference. The boxes marked the most relevant changes within the <i>ompk36</i> sequences. Alterations are described above the boxes and the positions of the corresponding amino acid residues are indicated below the boxes.</p