Advanced Amperometric Respiration Assay for Antimicrobial Susceptibility Testing

A ferricyanide-based electrochemical cell respiration assay was adapted for use in broad-spectrum antimicrobial susceptibility testing (AST). Total bacterial respiration was converted into faradaic current by electro-oxidation of ferrocyanide, produced when ferricyanide is reduced by bacterial electron-transport. For Escherichia coli (E. coli), the signal was linear with 5–13 × 10⁸ colony-forming units in measuring buffer. For AST, test cells were treated with drugs before ferricyanide addition; cell counts from the amperometric assay provided a measure of drug-induced cell death. Initial trials with six antimicrobial agents produced incorrect susceptibility classifications for drugs that were electroactive at the potential used to detect ferrocyanide or which affected cellular respiration rates. We therefore changed the procedure from drug-treatment and assay in the same buffer to sequential drug exposure in treatment buffer, centrifugal separation of surviving cells, cell resuspension, incubation in the presence of ferricyanide and finally ferrocyanide amperometry in drug-free buffer. Data analysis with E. coli led to an activity classification that agreed with cell culture-based ASTs, obtained by a quicker, more convenient procedure. The potential of this approach was confirmed by trials with the highly virulent bacterium Burkholderia pseudomallei, a particularly antimicrobial-resistant pathogen that is the cause of lethal melioidosis in tropical climates and is currently of concern as a potential bioterrorism agent

Swelling of <i>Bps</i>Omp38 proteoliposomes induced by cephalosporins and carbapenems.

<p>The proteoliposomes were prepared at pH–6 independent experiments. Levels of <i>Bps</i> resistance were determined by a broth micro dilution assay as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095918#pone-0095918-t001" target="_blank">Table 1</a>. R represents resistant; S, sensitive; and I, intermediate. Differences in the three data sets were evaluated using one-way ANOVA. Statistically significant differences (P<0.05) are marked with an asterisk (*). Values are as means ± SD, obtained from 4–6 independent sets of experiments.</p

Summary of relative permeabilities of <i>Bps</i>Omp38 and the levels of <i>Bps</i> resistance to cephalosporin and carbapenem, together with the structures of the drugs.

<p>The value in brackets is charge state of each antibiotic at pH<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095918#pone.0095918-Irwin1" target="_blank">[35]</a>.</p

A ribbon representation of the homology model of <i>Bps</i>Omp38.

<p>The modeled structure of <i>Bps</i>Omp38WT (A) shows the key residue Y119 (red) protruding into the channel lumen (top view). This residue was mutated to Ala or Phe, generating two single mutants, <i>Bps</i>Omp38Y119A (B) and <i>Bps</i>Omp38Y119F (C).</p

Purification and immunodetection of the <i>Bps</i>Omp38 variants, expressed in <i>E. coli</i>.

<p>(A) Chromatographic profile of <i>Bps</i>Omp38 purification with a Hitrap SP HP column, connected to an ÄKTA Prime plus FPLC system. The protein was eluted with a linear gradient of 0–1 M KCl, (B) SDS/PAGE followed by Coomassie Blue staining, (C) immunoblot analysis of the same samples as in panel B, using polyclonal anti-<i>Bps</i>Omp38 serum, (D) immunoblot analysis using anti-His₆ monoclonal antibody, and (E) identification of tryptic digests of the expressed proteins by nanoLC/ESI MS (First BASE Laboratories, Malaysia). Eight peptides, designated P1–P8 and shown in red, were unambiguously identified by MASCOT as identical to internal sequences of <i>Bps</i>Omp38. The 20-aminoacid signal peptide is underlined. Lanes: Std, standard proteins; 1, <i>Bps</i>Omp38WT; 2, <i>Bps</i>Omp38Y119A; 3, <i>Bps</i>Omp38 mY119F; 4, refolded <i>Bps</i>Omp38; 5, <i>E. coli</i> OmpF; 6, <i>E. coli</i> OmpN; 7, and <i>V. harveyi</i> ChiP.</p

Ion current recordings obtained by the black lipid membrane (BLM) reconstitution technique.

<p>BLM measurements of successive insertions of <i>Bps</i>Omp38 trimers at a transmembrane potential of +100 mV. Lipid bilayers were formed across a 200-µM aperture by the ‘painting’ technique using 50 mg.mL⁻¹ azolectin in <i>n</i>-hexane and bathed on either side in 1 M KCl. <i>Bps</i>Omp38 (1 µg.mL⁻¹) was added on the <i>cis</i> side. (A) <i>Bps</i>Omp38WT, (B) <i>Bps</i>Omp38Y119A, and (C) <i>Bps</i>Omp38Y119F. Left panels are ion current traces acquired for 100 s. Fast insertion of one <i>Bps</i>Omp38WT channel, occurring within the millisecond time-resolution, was captured and shown as an inset. The traces represent multiple insertions of <i>Bps</i>Omp38 variants produced by applied membrane potentials of +100 mV. Right panels are histograms from the corresponding traces, giving the probability of a pore conductance (G) averaged over several hundred inserting channels as indicated. The black line represents a single Gaussian fit.</p

Ion current recordings obtained by the black lipid membrane (BLM) reconstitution technique.

<p>BLM measurements of successive insertions of <i>Bps</i>Omp38 trimers at a transmembrane potential of +100 mV. Lipid bilayers were formed across a 200-µM aperture by the ‘painting’ technique using 50 mg.mL⁻¹ azolectin in <i>n</i>-hexane and bathed on either side in 1 M KCl. <i>Bps</i>Omp38 (1 µg.mL⁻¹) was added on the <i>cis</i> side. (A) <i>Bps</i>Omp38WT, (B) <i>Bps</i>Omp38Y119A, and (C) <i>Bps</i>Omp38Y119F. Left panels are ion current traces acquired for 100 s. Fast insertion of one <i>Bps</i>Omp38WT channel, occurring within the millisecond time-resolution, was captured and shown as an inset. The traces represent multiple insertions of <i>Bps</i>Omp38 variants produced by applied membrane potentials of +100 mV. Right panels are histograms from the corresponding traces, giving the probability of a pore conductance (G) averaged over several hundred inserting channels as indicated. The black line represents a single Gaussian fit.</p

Single channel recordings of <i>Bps</i>Omp38 porin in artificial lipid membranes.

<p>Typical ion current traces of single <i>Bps</i>Omp38 variant channels in a fully open state at a transmembrane potential of +100 mM, (B) Current-voltage relationship of <i>Bps</i>Omp38WT in comparison with its two mutants. The current through a single open <i>Bps</i>Omp38 channel was monitored in 1 M KCl, following discrete changes in the voltage across the phospholipid membrane, from −100 to +125 mV. The slopes of a linear fit yielded the single channel conductances of individual <i>Bps</i>Omp38 channels. Data points for <i>Bps</i>Omp38WT, <i>Bps</i>OmpY119A and <i>Bps</i>OmpY119F are plotted as circles, squares and triangles, respectively. Differences in the three data sets were evaluated using one-way ANOVA. Statistically significant difference (P<0.05) is shown with an asterisk (*).</p

Statistical analysis of MIC values of <i>Bps</i> shown in Table 1 by one-way ANOVA and log₂ dilution methods.

a<p>N is the total number of samples used for both analyses. Two equal-sized sampling groups (each group, n = 6) were used, in the absence and presence of PAβN.</p>b<p>NS represents non-significant difference between the two studied groups at <i>p</i><0.05.</p>c<p>NS represents non-significant difference between the two studied groups at essential agreement ≥ 85% <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095918#pone.0095918-Marley1" target="_blank">[37]</a>.</p

Swelling of <i>Bps</i>Omp38-containing proteoliposomes, induced by neutral sugars.

<p>For each preparation, multilamellar liposomes were reconstituted with 200<i>Bps</i>Omp38. The isotonic concentration was defined as the concentration of <i>D</i>-raffinose that caused no change in the absorbance at 500 nm of the proteoliposome suspension, over a period of 60 s. (A) Permeation of different types of sugars through <i>Bps</i>Omp38 reconstituted liposomes. Each swelling rate was normalized to the rate of swelling in arabinose, which was set to 100%. (B) Semilogarithmic plot of relative permeation rates of sugars through the proteoliposomes reconstituted with <i>Bps</i>Omp38WT and other mutants. The logarithm of the permeation rate is plotted against the molecular weight of the sugar. Differences in the three data sets were evaluated using one-way ANOVA. Statistically significant differences (P<0.05) are marked with an asterisk (*). Values are as means ± SD, obtained from 4–6 independent sets of experiments.</p