Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa

MICs were determined by broth microdilution following the guidelines from the Clinical and Laboratory Standards Institute and using P. aeruginosa ATCC27853 as a quality control. Susceptibility was assessed according to the interpretive criteria of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or using a cut-off value of 16 mg/L for temocillin (no EUCAST breakpoint) by analogy with the current susceptibility breakpoint of its parent compound ticarcillin. For specific isolates, MICs were also measured in the presence of 20 mg/L Phe-Arg-β-naphthylamide dihydrochloride (PAβN, broad-spectrum efflux pump inhibitor10 (link); Sigma-Aldrich) and of 1 mM MgSO₄ (to prevent the outer membrane permeabilization caused by PAβN33 (link)) or in the presence of substrates of P. aeruginosa porins, namely vanillate (VNL, OpdK substrate18 (link)), or L-arginine (L-Arg, OprD substrate19 (link)), both obtained from Sigma-Aldrich.

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Chalhoub H., Pletzer D., Weingart H., Braun Y., Tunney M.M., Elborn J.S., Rodriguez-Villalobos H., Plésiat P., Kahl B.C., Denis O., Winterhalter M., Tulkens P.M, & Van Bambeke F. (2017). Mechanisms of intrinsic resistance and acquired susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients to temocillin, a revived antibiotic. Scientific Reports, 7, 40208.

Publication 2017

MgSO4 vanillate L-arginine

Aeruginosa p Antimicrobial Clinical laboratory European L arg Membrane Mgso4 Mics Parent Phe arg naphthylamide Porins Susceptibility Temocillin Ticarcillin

Corresponding Organization :

Other organizations : Queen's University Belfast, Cliniques Universitaires Saint-Luc, Université de franche-comté, Centre Hospitalier Universitaire de Besançon, University Hospital Münster, Erasmus Hospital

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Variable analysis

independent variables

Presence of 20 mg/L Phe-Arg-β-naphthylamide dihydrochloride (PAβN, broad-spectrum efflux pump inhibitor)
Presence of 1 mM MgSO4 (to prevent the outer membrane permeabilization caused by PAβN)
Presence of vanillate (VNL, OpdK substrate)
Presence of L-arginine (L-Arg, OprD substrate)

dependent variables

Minimum inhibitory concentrations (MICs)

control variables

Broth microdilution method following the guidelines from the Clinical and Laboratory Standards Institute
Using P. aeruginosa ATCC27853 as a quality control
Susceptibility assessed according to the interpretive criteria of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or using a cut-off value of 16 mg/L for temocillin

controls

Positive control: P. aeruginosa ATCC27853
No negative control explicitly mentioned

Annotations

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