MICs of cefoxitin and amoxicillin tested alone and with PAβN, cloxacillin and clavulanic acid towards 4 representative <i>K. pneumoniae</i> strains.

<p>Mol: molecule, FOX: cefoxitin, AMX: amoxicillin, CLX: cloxacillin, CLA: clavulanic acid, *: 0.096 mM, ^♦: 0.117 mM, ^Δ: 2 mg/L, ^†: 20 mg/L.</p

MICs of cefoxitin and nalidixic acid tested alone and with inhibitor efflux (PAβN) and sub-inhibitory concentrations of cloxacillin towards 4 representative <i>K. pneumoniae</i> strains.

<p>FOX: cefoxitin, NAL: nalidixic acid, CLX: cloxacillin, *: 0.096 mM (50 mg/L), ^♦: 1/20 CLX MIC.</p

Scheme of the selective efflux of ß-lactam molecules and the effect of various molecules.

<p>A: the AcrAB-TolC efflux pump. B: the FOX efflux. C: the effect of PAßN on FOX efflux. D: the effect of PAßN+CLX on FOX efflux. OM, outer membrane; P, periplasmic space; IM, inner membrane; PBP, penicillin binding protein (ß-lactam target). Cloxacillin (CLX) and cefoxitin (FOX) are represented by black squares or black circles respectively; and grey triangles represent the PAßN. Black straight arrows represent the drug penetration through the outer membrane and the black curved lines represent the drug efflux through the efflux pumps. Bold and dotted curved lines indicated the high and low level of FOX efflux respectively. Empty circles and triangles represent the ß-lactam selective sites and the non-selective sites, respectively. PAßN is able to bind to the non-selective site located inside the pump cavity and at a lesser extent (due to affinity) to the ß-lactam site. In the presence of ß-lactam (CLX or FOX), the ß-lactam site is preferentially occupied by the ß-lactam molecules. For clarity reasons, only the first hypothesis was presented. For the second hypothesis (two pumps), the different drug affinity sites (non-selective and ß-lactam selective sites) may be distributed in the two efflux pumps acting in the same time in resistant strains.</p

Detection of AcrA and TolC in <i>Klebsiella pneumoniae</i> isolates.

<p>Top part, immunodetection was carried out with antiserum directed against denatured AcrA; bottom part, immunodetection was carried out with antiserum directed against denatured TolC porin. WT, <i>E. aerogenes</i> wild type strain producing normal level of AcrA and TolC; <i>acrA</i>, <i>acrA</i> deleted strain; <i>tolC</i>, <i>tolC</i> deleted strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004817#pone.0004817-Pradel1" target="_blank">[16]</a>. a and b indicate the migration of the molecular weight marker 30 kD and 43 kD, respectively. ATCC, ATCC11296, lanes 1 to 11, strain KPBj 1 to strain KPBj 11.</p

MICs of various antibiotics tested alone and with efflux inhibitor PAβN* towards 11 <i>K. pneumoniae</i> clinical isolates and ATCC strains.

<p>*: 0.096 mM (50 mg/L), CMP: chloramphenicol, NAL: nalidixic acid, OFX: ofloxacin, AMX: amoxicillin, AMC: amoxicillin+clavulanic acid, PIP: piperacillin, TZP: piperacillin+tazobactam, FOX: cefoxitin, CAZ: ceftazidime, FEP: cefepime, ERT: ertapenem, CLX: cloxacillin, ERY: erythromicin, +: with PAβN, −: without PAβN, nd: not determined.</p

MICs of cefoxitin for two <i>K. pneumoniae</i> isolates (KpBj 7 and 9) and strain ATCC 11296 measured in the presence of efflux inhibitor PAβN (0.096 mM) and increased concentrations of cloxacillin (CLX).

<p>MIC percent (MIC%) represents the decrease in cefoxitin MIC in the presence of different CLX concentrations (plotted as CLX (mM)) compared with MIC measured without PAßN and CLX.</p

Clinical and molecular characters of 11 <i>K. pneumoniae</i> clinical isolates less susceptible to cefoxitin and susceptible to amoxicillin+clavulanic acid.

<p>ICU: intensive care unit.</p

Cloxacillin dose-effect on MICs of nalidixic acid tested alone and with PAβN towards 4 representative <i>K. pneumoniae</i> strains.

<p>NAL: nalidixic acid, *: 0.096 mM (50 mg/L), CLX: cloxacillin.</p

Detection of porins in <i>Klebsiella pneumoniae</i> isolates.

<p>Top part, immunodetection was carried out with antiserum directed against denatured OmpF porin; bottom part, immunodetection was carried out with antiserum directed against denatured OmpC porin. In the two incubation assays, antiserum directed against denatured OmpA was used as control. ATCC: ATCC11296; lane 1 to 11, strain KPBj 1 to strain KPBj 11; OmpF and OmpC illustrate the migration of the <i>E. coli</i> porins in the same conditions (used as internal standards in SDS-PAGE). Arrows and circles indicate the migration of porins and OmpA, respectively; a, indicates the migration of the molecular weight marker (30 kD).</p

Additional file 2: Table S2. of The ST131 Escherichia coli H22 subclone from human intestinal microbiota: Comparison of genomic and phenotypic traits with those of the globally successful H30 subclone

Escherichia coli virulence factor encoding genes tested by the PCR method. (DOCX 25 kb