Conventional microbiology versus PCR/ESI-MS test results.

#<p>Level of Detection– Reported as genome equivalents per PCR reaction (GE/well).</p>*<p>LLL  =  Left lower lobe; RLL  =  Right lower lobe; ET aspirate  =  endotracheal aspirate.</p>#<p>CSF  =  cerebrospinal fluid; BAL fluid  =  bronchoalveolar lavage fluid.</p

(A) Antibiograms of <i>P. aeruginosa</i> PAO pΔEP-SPM-1 and <i>P. aeruginosa</i> 48-1997A against disks embedded with 30 µg ceftazidime (CAZ), 30 µg cefepime (CFP) and 10 µg imipenem (IMI), alone or faced to 1.5 mg dipicolinic acid (DPA) containing disks.

<p>Halos of inhibition are remarked with dotted lines. (B) Ceftazidime hydrolysis rates (300 µM) of crude extracts of <i>P. aeruginosa</i> PAO pΔEP-SPM-1 and <i>P. aeruginosa</i> 48-1997A, with and without 20 mM DPA. Reaction medium was 10 mM Tris, 30 mM MgCl₂ at pH 7.3.</p

Values of <i>Tm_app</i> for different SPM-1 mutants and their apo derivatives, measured in periplasmic extracts of <i>P. aeruginosa</i> PAO by β-lactam hydrolysis rates and protein solubility.

<p>^ndNot determined.</p><p>^*The apo derivatives of these mutants precipitated during metal removal.</p

Simplified scheme of the H-bridge interaction network present in the different variants of SPM-1 under study.

<p>Holo and apo-like conformations are highlighted in blue and yellow, respectively.</p

Minimum inhibitory concentration (MIC) of imipenem (IMI), piperacillin (PCL), ceftazidime (CAZ), cefoxitin (CXT) and cefepime (CFP) for selected strains of <i>P. aeruginosa</i> (producing the different SPM-1 mutants at positions 84 and 121).

<p>No SPM-1 = <i>P. aeruginosa</i> containing the vector pBBR-MCS5. Values come from 5 independent determinations.</p

Patients, specimens, and antimicrobial treatment.

*<p><b>DOT</b>– Days of therapy; <b>POD</b> – Post-operative Day; <b>S/P</b> – Status-post; <b>LP</b> – lumbar puncture; <b>I&D</b> – incision and drainage.</p><p><b>MSSA</b> – Methicillin susceptible <i>Staphylococcus aureus</i><b>; CBD</b> – Common bile duct; <b>TMP/SMX</b> – Trimethoprim-Sulfamethoxazole.</p><p><b>RLE/LLE</b> – right/left lower extremity; <b>TKA</b>– Total Knee Arthroplasty; <b>ORIF</b>– Open Reduction and Internal Fixation.</p><p><b>UTI</b> – Urinary tract infection; <b>AVN</b> – avascular necrosis; <b>IE</b> – infective endocarditis; <b>AV</b> – aortic valve; <b>MV</b> – mitral valve.</p><p><b>CAP</b>–Community acquired pneumonia; <b>VAP</b>–Ventilator associated pneumonia; <b>HCAP</b>–Healthcare associated pneumonia.</p><p><b>BAL</b> – bronchoalveolar lavage; <b>RLL/LLL</b> – right/left lower lobe; <b>ET</b> – endotracheal tube; <b>ARDS</b> – acute respiratory distress syndrome.</p

β-lactamase activity of SPM-1 mutants in <i>P. aeruginosa</i> periplasmic extracts against cephalotin (CFL), cephalexin (CFX), ceftazidime (CAZ), cefepime (CFP) and imipenem (IMI).

<p>Rates of hydrolysis are normalized to the amount of periplasmic protein amounts (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003817#s4" target="_blank">Materials and Methods</a>). Reactions were accomplished in Tris 10 mM, MgCl₂ 30 mM pH 7.3 and 800 µM antibiotic. Error bars come from activity measurements from five independent periplasmic fractionations.</p

MIC values against cefepime of <i>P. aeruginosa</i> PAO containing SPM-1 mutants at different concentrations of the metal chelator dipicolinic acid (DPA).

<p>MIC values against cefepime of <i>P. aeruginosa</i> PAO containing SPM-1 mutants at different concentrations of the metal chelator dipicolinic acid (DPA).</p

(A) Location of first shell (green) and second shell (orange) residues in the structure of SPM-1.

<p><b>(B)</b> Schematic representation of the active sites of the B1 MβLs NDM-1 and SPM-1 emphasizing the differences in the hydrogen bond networks involving metal ligands (H116, H118, H196, D120, C221 and H263, in green) and second sphere residues (115, 84, 121, 69, 70 and 262, in orange) due to residues 84 (D or S) and 121 (K or G).</p

Residues present at positions 84 and 121 in selected mutants for each antibiotic selection and library in <i>P. aeruginosa</i> PAO.

<p>IMI = imipenem, PCL = piperacillin, CAZ = ceftazidime, CXT = cefoxitin.</p