Additional file 1 of Efficacy of single antibiotic therapy versus antibiotic combination in implant-free staphylococcal post-surgical spinal infections: a retrospective observational study

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Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of l,d‑Transpeptidase Ldt_Mt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by β‑Lactamase BlaC

Combinations of β-lactams of the carbapenem class, such as meropenem, with clavulanate, a β-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual β-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum β-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the β-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide–alkyne Huisgen cycloaddition reaction catalyzed by copper­(I) is fully compatible with the highly unstable β-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the l,d-transpeptidase Ldt_Mt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of Ldt_Mt1 and reduced hydrolysis by BlaC

Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of l,d‑Transpeptidase Ldt_Mt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by β‑Lactamase BlaC

Combinations of β-lactams of the carbapenem class, such as meropenem, with clavulanate, a β-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual β-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum β-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the β-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide–alkyne Huisgen cycloaddition reaction catalyzed by copper­(I) is fully compatible with the highly unstable β-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the l,d-transpeptidase Ldt_Mt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of Ldt_Mt1 and reduced hydrolysis by BlaC

Determination of turnover numbers for full catalytic cycles leading to hydrolysis of β-lactams by Ldt_fm.

<p>Turnover numbers were determined for hydrolysis of ceftriaxone (A) and ampicillin (B) by Ldt_fm (5 µM).</p

Average mass of acylenzymes (mass increment)^a.

a<p>The mass increment was calculated by subtracting the mass of the native enzyme (29,009.3) from the mass of acylenzymes.</p><p>NA, not applicable.</p

Chemical shift perturbations induced by non-covalent binding of β-lactams to Ldt_fm C442A.

<p>Chemical shift perturbations (CSPs) of Ldt_fmC442A residues are reported as a function of the antibiotic to protein molar ratio. Closed square, Lys394 for ertapenem; closed triangle, Trp385 for ceftriaxone; grey circle, Ser423 for ampicillin. The end point of the titration was determined by the solubility limit of the antibiotics. Experimental data were fitted (solid lines) with equation 2 described in the experimental procedures. <i>K</i>_D values of 50, 44, and 79, mM were determined for binding of ertapenem, ceftriaxone, and ampicillin to Ldt_fmC442A, respectively. Ertapenem was used as a representative of the carbapenem family since the low solubility of imipenem precluded <i>K</i>_D determination for this antibiotic.</p

Mass spectrometry analysis of kinetics of Ldt_fm inactivation by β-lactams.

<p>Ldt_fm (20 µM) was incubated with 200 µM of β-lactams. Left panels, representative mass spectra obtained after 0.3, 5, and 10 min of incubation of Ldt_fm with indicated β-lactams. Pair of peaks labeled with the same letter are [M+32H]³²⁺ and [M+31H]³¹⁺ ions. (A) imipenem, peaks a and a’ at <i>m</i>/<i>z</i> 916.93 and 946.48 correspond to acylenzyme EI*. (B) Ceftriaxone, peaks a and a’ at <i>m</i>/<i>z</i> 907.58 and 936.84 correspond to free enzyme. Peaks b and b’ at <i>m</i>/<i>z</i> 919.95 and 946.60 correspond to acylenzyme EI*. (C) Ampicillin, peaks a and a’ (<i>m</i>/<i>z</i> 907.52 and 936.79), b and b’ (<i>m</i>/<i>z</i> 918.52 and 948.08), and c and c’ (<i>m</i>/<i>z</i> 913.51 and 942.95) correspond to free enzyme, EI*, and EI**, respectively. Right panels, kinetics of Ldt_fm-β-lactam adducts formation. Relative intensities were deduced from peak heights. Blue diamond, free enzyme; Red square EI*; Green triangle EI**.</p

Inactivation of <i>E. faecium</i> L,D-transpeptidase (Ldt_fm) by β-lactams.

<p>Reaction schemes for Ldt_fm inactivation by β-lactams of the carbapenem (imipenem), cephem (ceftriaxone), and penam (ampicillin) classes. E, free form of the enzyme; EI^ox, oxyanion; EI* and EI**, acylenzymes. SH, sulfhydryl of the catalytic cysteine.</p

Kinetics of Ldt_fm inactivation by imipenem, ceftriaxone, and ampicillin.

<p>Fluorescence kinetic data were acquired with a stopped-flow apparatus. Trp residues of Ldt_fm were excited at 224 nm and fluorescence emission was determined at 335 nm to monitor quenching upon β-lactam binding. Kinetics were biphasic for imipenem (A) providing estimates of catalytic constants <i>k</i>₁, <i>k</i>₋₁, and <i>k</i>₂(B). See Supplementary methods in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067831#pone.0067831.s001" target="_blank">File S1</a> for the iterative fitting method and Supplementary Fig. S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067831#pone.0067831.s001" target="_blank">File S1</a> for the complete set of data. Monophasic fluorescence decreases observed for ceftriaxone (C) and ampicillin (D) were fitted to exponential decays (representative plots are shown). Regression analysis was performed with equation F_t = F_eq+ΔF e^{−<i>k</i>obst} in which F_eq and F_t are the fluorescence intensities at equilibrium and at time t, respectively, ΔF is the difference between fluorescence intensity at time = 0 and at equilibrium, t is time, and <i>k</i>_obs is a constant. The resulting rate constants (<i>k</i>_obs) increased linearly with the drug concentration (E) and the slope provided an estimate of the efficiency of enzyme acylation (F).</p

Determination of ampicillin-free Ldt_fm using rapid inactivation by imipenem.

<p>(A) Ldt_fm (20 µM) was incubated with ampicillin (200 µM) for indicated time and imipenem was used to determine the concentration of free enzyme by using stopped-flow spectrophotometry at 299 nm. The concentration of free Ldt_fm reached a plateau revealing equilibrium between the various enzyme forms. The concentration of free Ldt_fm slowly increased after 100 min due to a decrease in ampicillin concentration. (B) Concentration of free Ldt_fm at equilibrium as a function of ampicillin concentration. Data are mean ±SD of 3 experiments.</p