The Activity of 16 New Hydantoin Compounds on the Intrinsic and Overexpressed Efflux Pump System of Staphylococcus aureus

Materials and Methods

Compounds and materials. Sixteen hydantoin derivatives were synthesized by methods to be presented elsewhere and were evaluated for inhibitory effects on the efflux pump systems of S. aureus. The hydantoin compounds and their structures are listed in Table I. Each contained two aromatic rings and were divided into four groups (A-C and MF8) according to the position of each aromatic fragment. Group A comprised 11 phenylpiperazine 5,5-dimethylhydantoin derivatives (PI1a-PI11a) possessing two terminal (un)substituted phenyl rings within substituents at N1 and N3 positions of the hydantoin. Group B included two racemic N3-ester derivatives of 5,5-diphenylhydantoin with 2-hydroxypropyl linker between hydantoin and hydroxyethylpiperazine (KF4) or morpholine (KF20). Two 5-arylidene derivatives (HY83 and HY84), possessing two benzene moieties connected to each other by ether linker, belonged to group C. Aromatic rings of compound MF8 are found at position 5 and at the terminal part of the aminealkyl substituent at position N3 of the hydantoin structure. Syntheses of compounds KF4, KF20, HY83 and HY84 have been described elsewhere (10, 11). Syntheses of compounds PI1a-PI11a and MF8 involved two different pathways and will be described elsewhere. Purity and identity of the 15 hydantoin derivatives were confirmed by the use of spectroscopic methods [nuclear magnetic resonance spectroscopy (¹H-NMR), infrared spectroscopy (IR), elemental analysis, melting point measurement and thin-layer chromatography (TLC)].

The compounds were dissolved in dimethyl sulfoxide (DMSO). Ethidium bromide (EB) was purchased from Sigma (Madrid, Spain). Tryptic soy in powder form was purchased from Difco (Madrid, Spain) for the preparation of broth and agar.

Bacteria. Wild-type Staphylococcus aureus American Type Culture Collection (ATCC) 25923 and the HPV-107 MRSA strain (representative of the MRSA Iberian clone, was isolated at a Portuguese hospital in 1992 and is characterised by resistance to several classes of antibiotics, particularly β-lactams, aminoglycosides, fluoroquinolones, macrolides, rifampicin and tetracycline) whose partial resistance to oxacillin is due the presence of a plasmid that contains the gene coding for the Qac efflux pump (9). The HPV-107 strain was graciously provided by Professor Dr. H. De Lencastre.

Determination of minimum inhibitory concentrations (MICs). The MICs of compounds, including EB, were determined by using broth microdilution method in Mueller-Hinton Broth (MHB), according to Clinical and Laboratory Standards Institute (CLSI) recommendations (12). The MICs were required in order that the concentrations of each hydantoin used in the study would be no greater than ½ or ¼ of its MIC, and hence, would not affect the viability of the bacteria (13, 14).

Real-time EB accumulation assay. The activity of compounds on the real-time accumulation of EB was assessed by the automated EB method, previously described in detail elsewhere (13, 14), using a Rotor-Gene 3000™ thermocycler with real-time analysis software (Corbett Research, Australia). Briefly, strains were cultured in tryptic soy broth (TSB) medium until they reached an optical density (OD) of 0.6 at 600 nm; they were then centrifuged at 10000× g for 3 minutes, the pellets were re-suspended in phosphate-buffered saline (PBS) (pH 7.4) with a final concentration of glucose of 0.4% and the OD adjusted to 0.6 at 600 nm. Aliquots of 45 μl of the cell suspension were distributed to 0.2 ml tubes. The compounds were individually added at different concentrations (50 and 200 mg/l final concentration) in 5 μl volumes of their stock solutions, and finally 45 μl of EB was added to yield a final concentration of 1 mg/l (Sigma-Aldrich Quimica SA, Madrid, Spain) in PBS with glucose. It is important to note that prior to the experiments described, the maximum concentration of EB which was within the capacity of the bacterium to extrude was determined and shown to be exactly that previously reported (13, 14). For the wild-type reference bacterial strain employed in the study, this concentration of EB was determined to be 0.25 mg/l (9). The tubes were placed into a Rotor-Gene 3000™ thermocycler and the fluorescence monitored on a real-time basis.

From the real-time data, the activity of the compound, namely the relative final fluorescence (RFF) of the last time point (minute 20) of the EB accumulation assay was calculated according to the formula: where RF_treated is the relative fluorescence (RF) at the last time point of the EB retention curve in the presence of an inhibitor; RF_untreated is the relative fluorescence at the last time point of the EB retention curve of the untreated control having the solvent control (DMSO). The greater the difference between RF_treated and RF_untreated, the greater the degree of the EB accumulated and, therefore, the greater the degree of inhibition of the efflux pump system of the bacterium by the hydantoin compound. Dividing the RFI by the number of micromoles of the hydantoin compound used in the assay provides a specific activity of inhibition of the efflux pump of the bacterium. Because the overexpressed efflux pump system is due to an increase of efflux pumps, the amount of hydantoin needed to inhibit the efflux pump system is greater than that needed to inhibit the intrinsic efflux pump of the wild-type strain.

Determination of the reduction of resistance to oxacillin by compounds that inhibit the efflux pump of the ATCC and HPV-107 S. aureus strains. The MIC of oxacillin was first determined with the aid of the microplate broth dilution method (12). The MIC for oxacillin was then assessed in the absence and presence of concentrations of the compounds that significantly inhibited the efflux pump (i.e. produced accumulation of EB). Reduction of resistance to oxacillin was considered to be significant when the MIC of oxacillin was reduced by at least four-fold.