COSA JUZGADA PRINCIPIO DE SEGURIDAD JURÍDICA

2.6.1. BacLight™ assay of membrane integrity

Bacterial membrane integrity following exposure to antibacterial agents was assessed using the Live/Dead BacLight™ bacterial viability kit (Life Technologies, Paisley, UK) as described previously (Hilliard et al., 1999). Briefly, S. aureus SH1000 was grown in MHB to an OD600 of 0.5 units, and volumes of culture (500 l) were washed twice and resuspended in 900 l of H2O. Volumes (100 l) of antibacterial compounds at a concentration of 40x MIC were then added and samples mixed for 10 minutes at 37oC. Following incubation, samples were washed twice and resuspended in 1 ml of H2O. In a dark room, 50 l of each sample were added to the wells of a blacked out 96-well microtitre plate containing 150 l of BacLight™ reagent (consisting of propidium iodide and SYTO 9 stock solutions diluted 300-fold in H2O) and incubated in the dark at room temperature for 15 min. Fluorescence was measured in a Fluostar Optima plate reader with excitation set at 485 nm and emission at 645 nm (propidium iodide fluorescence) or 530 nm (SYTO 9 fluorescence). Fluorescence values were normalised against drug-free controls and percentage membrane integrity was then expressed as the ratio of SYTO 9:Propidium iodide fluorescence relative to cultures treated with 0.5% (w/v) SDS.

Bacterial membrane integrity following exposure to antibacterial agents under anaerobic conditions was carried out as described above with the exception that all work was carried out in an MG1000 anaerobic workstation using MHB and sterile H2O that had been de-oxygenated for at least 16 h.

2.6.2. Membrane potential

Bacterial membrane potential following exposure to AgNO3 and comparator agents was measured over a 3 h time course using the fluorescent dye 3,3‘-dipropylthiadicarbocyanine iodide (DiSC3(5)) (Life Technologies, Paisley, UK) according to methods described previously (Hobbs et al., 2008), with adaptations. Overnight cultures of S. aureus SH1000 in MHB were diluted 1:100 into 20 ml of MHB and incubated at 37oC with aeration to an OD600 of 0.2 units. Cells were then pelleted by centrifugation (5000 x g, 15 min, 37oC), washed twice and resuspended in 20 ml of 5 mM HEPES, 5 mM glucose buffer (pH 7.0) containing 100 mM KCl and 2 M DiSC3(5) reagent. Cultures were then incubated as described above for an additional 30 min in the dark before addition of test compounds to a final concentration of 4X MIC. Cultures continued to be incubated for 120 min with samples taken at 0, 10, 20, 30, 40, 50, 60, 90 and 120 min.

Samples removed at time-points were centrifuged at 13,000 x g for 30 sec and the supernatant (containing extracellular dye) transferred into an equal volume of DMSO. Cell pellets were re-suspended in DMSO and incubated at room temperature for 10 min to release intracellular dye prior to the addition of an equal volume of 5 mM HEPES, 5 mM glucose buffer (pH 7.2). The fluorescence of both extracellular and intracellular samples was measured in a Perkin Elmer LS 45 luminescence spectrometer with excitation set at 622 nm and emission at 670 nm. The membrane potential at each time point was then calculated using the Nernst equation (see below) and then expressed as a percentage of the time 0 value.

( ) ( )

Where R is the gas constant (8.3144621), T is the temperature in degrees Kelvin, F is the Faraday constant and If and Ef are the intracellular fluorescence and extracellular fluorescence respectively.

2.6.3. Construction of liposomes

Carboxyfluorescein-filled liposomes were constructed as described previously (StGelais

et al., 2007), with modifications. Stock solutions (10 mg/ml in chloroform) of

L--phosphatidic acid (PA), L--phosphatidylcholine and lissamine rhodamine B-labelled L--phosphatidylethanolamine (RPE) were mixed in a 49:49:2 ratio to a final volume of 100 l in a glass test tube. All lipids were purchased from Avanti polar lipids (Alabaster, Alabama, USA). All chloroform was removed by first drying the lipids under a stream of argon then placing the tubes under vacuum for 2 h prior to adding 1 ml of CF buffer (50 mM carboxyfluorescein, 10 mM NaCl, 10 mM HEPES, 134 mMNaOH, pH 7.4) and incubating overnight at 30oC with shaking (200 rpm). Liposomes were produced by extrusion through a 0.4 m filter in an Avanti mini-extruder with glass syringes and purified by ultracentrifugation (100,000 x g, 20 min) and washing three times in assay buffer (1 mM HEPES, 107 mM NaCl, pH 7.4). Liposomes were resuspended in 500 l of assay buffer and the liposome concentration calculated by measuring rhodamine absorbance at OD570. To create ―staphylococcal‖ liposomes, this method was repeated, but the lipids described above were substituted for 1,2-dioleoyl-sn-phosphatidylglycerol (PG), cardiolipin (CL) and RPE in a 59:39:2 ratio (Epand, et al., 2007). Liposomes of E. coli membrane phospholipid were created using E. coli polar lipid extract (consisting 67% [w/w] phosphatidylenanolamine, 23.2% [w/w] PG and 9.8 % [w/w] CL) mixed with RPE in a 48:2 ratio.

2.6.4. Liposome damage assay

To the wells of a blacked-out 96-well plate, 50 mM of liposomes were mixed with the antibacterial agent to be tested at 4x MIC and incubated in the dark at 37oC for 2 h. Fluorescence measurements were then taken in a Fluostar Optima plate reader with excitation at 485 nm and emission at 520 nm. Fluorescence values were normalised to drug-free controls and the percentage liposome integrity expressed as the fluorescence intensity relative to that of liposomes treated with 0.1% (w/v) Triton X-100.

2.6.5. -galactosidase leakage

Assessment of -galactosidase leakage from S. aureus was carried out as described previously (O'Neill et al., 2004), with the exception that the assays were performed using strain SH1000. Briefly, cultures of SH1000 were grown in MHB to an OD600 of 0.6 units before the addition of AgNO3 and comparator compounds at a final concentration of 4X MIC. Following a 10 min incubation at 37 oC, cells were removed by centrifugation and 4-methylumbelliferyl--D-galactoside was added to samples (180 l) of culture supernatant (final concentration 1 mg/ml) prior to incubation for 2 h at 25 oC. Sample fluorescence was measured (excitation 365 nm, emission 460 nm) in a Fluostar Optima plate reader plate reader and values converted into fluorophore (4-methylumbelliferone) concentration using a calibration curve consisting of known fluorophore concentrations.

2.6.6. Bacillus subtilis reporters

Five B. subtilis antibiotic biosensors containing specific promoters that are induced by inhibition of DNA, RNA, fatty acid, cell wall and protein biosynthesis were utilised as described previously (Urban et al., 2007), with modifications. Strains were grown in

LBB to an OD600 of 0.2 units, then samples (90 l) of culture were treated with AgNO3 or an appropriate control antibiotic in a 96 well plate and incubated at 37oC for 1-3 h (depending on the construct). Volumes (60 l) of 0.8M luciferin in 0.1M citrate buffer (pH 5.0) were then added and the luminescence of the sample recorded by a Fluostar Optima plate reader. Luminescence values were compared relative to a non-induced control and a >2-fold increase in luminescence was considered a positive result.

2.6.7. Macromolecular synthesis assays

The inhibition of DNA, RNA, protein and peptidoglycan synthesis by AgNO3 and control compounds was monitored by determining the incorporation of radiolabelled precursors [methyl-3H] thymidine (DNA), [5, 6-3H] uridine (RNA), L-[G-3H] glutamine (protein) and [1-14C] glycine (peptidoglycan), as described previously (Cherrington et

al., 1990). All radiolabelled compounds were purchased from Perkin-Elmer

(Cambridge, UK). Overnight cultures of SH1000 in MHB were diluted 1:100 in 9 ml MHB and incubated at 37oC with aeration and shaking (200 rpm) to an OD600 of 0.2 units. Isotopes were added at a final concentration of 1 Ci/ml for 3H compounds and 0.1 Ci/ml for 14C-glycine 10 min prior to addition of antibacterial agent at a final concentration of 4X MIC. Following a further 10 min incubation, 500 l of culture was added to 4.5 ml of 10 % (w/v) ice-cold trichloroacetic acid (TCA) and incubated on ice for 40 min to precipitate macromolecules. Samples were then processed as previously described (Wilson et al., 1995) and radioactivity measured using a TriCarb 2100TR liquid scintillation counter (Packard Bioscience Ltd, Berkshire, UK).

2.6.8. Enzyme specificity assays

2.6.8.1. RNA polymerase

The ability of Ag+ and comparator compounds (at 4X MIC) to inhibit E. coli RNA polymerase (RNAP) was determined by measuring transcription using SYBR green as described previously (Mariner et al., 2010). Briefly, test compounds (or compound diluents) were mixed with 0.2 U of E. coli core RNAP (Epicentre, Madison, Wisconsin, USA) and incubated at 37oC for 10 min prior to the addition of KoolTM NC-45TM Universal RNA polymerase template (Epicentre) and rNTPs to a final concentration of 125 ng/ml and 0.5 mM respectively. Following incubation at 37oC for 2 h, the reaction was stopped by the addition of EDTA to a final concentration of 20 mM EDTA (pH 8) and the amount of transcript produced was measured by adding 0.07% (v/v) SYBR-Green I (Life Technologies) and determining fluorescence intensity at 520 nm, using an excitation wavelength of 497 nm. Fluorescence readings were determined using an EnVision 2103 Multilabel Reader (Perkin-Elmer). Percentage inhibition of RNAP by test compounds was calculated as the fluorescence intensity relative to reactions treated with compound diluents, following normalisation to template-free controls.

2.6.8.2. Malate dehydrogenase

The ability of Ag+ and comparator agents (at 4X MIC) to inhibit the conversion of oxaloacetic acid + -NADH to L-malic acid and -NAD by malate dehydrogenase was determined spectrophotometrically as described previously (Seidler et al., 2003). Briefly, test compound was added to a cuvette containing 50 mM KPO4 buffer (pH 7.35), followed by addition of malate dehydrogenase (final concentration 0.002 M), NADH (Boehringer Mannheim; final concentration 0.2 mM) and

oxaloacetic acid (final concentration 0.2 mM). The contents of the cuvette were mixed and OD340 readings were taken every 30 sec for 6.5 min in a SPECTRAmax PLUS384 Microplate Spectrophotometer (Molecular Devices Ltd, Sunnyvale, California, USA). Readings were plotted on a scatter graph in Microsoft Excel software and the rate of reaction calculated as the gradient of a trendline fitted to the data. Percentage inhibition was calculated using the following equation:

((

) )

Where Rc= Rate of reaction when exposed to antibacterial agent, Rb= Rate of reaction with no malate dehydrogenase present and Rd=Rate of reaction when exposed to diluent only.

2.6.8.3. Chymotrypsin

The ability of Ag+ and comparator agents (at 4X MIC) to inhibit the conversion of the substrate Suc-AAPF-pNA to 4-nitroaniline by chymotrypsin was determined spectrophotometrically as described previously (Seidler et al., 2003). Briefly, test compound was added to a cuvette containing 50 mM KPO4 buffer (pH7.35), followed by addition of chymotrypsin (final concentration 0.05 M) and Suc-AAPF-pNA (final concentration 0.15 mM). The contents of the cuvette were mixed and OD410 readings were taken every 30 sec for 6.5 min in a SPECTRAmax PLUS384 Microplate Spectrophotometer (Molecular Devices). Readings were plotted on a scatter graph in Microsoft Excel software and the rate of reaction calculated as the gradient of a trendline fitted to the data. Percentage inhibition was calculated using the equation described in section 2.6.8.2.

2.6.8.4. -galactosidase

The effect of Ag+ and comparator compounds (at 4X MIC) to inhibit the conversion of 4-methylumbelliferyl-β-D-galactopyranoside to 4-methylumbelliferone by

-galacotsidase was determined by fluorescence intensity. Test compound was added to the wells of a blacked-out 96-well plate containing AB buffer (100mM NaCl, 60mM K2PO4 and 40mM KH2PO4), followed by the addition of 1 U E. coli -galactosidase and 4-methylumbelliferyl-β-D-galactopyranoside to a final concentration of 1 g/ml. Reactions were incubated for 90 min at 25oC and the reaction stopped by addition of Na2CO3 to a final concentration of 0.2 M. Fluorescence intensity was measured in a Fluostar Optima plate reader at 460 nm, using an excitation wavelength of 365 nm. Percentage inhibition of -galactosidase by test compounds was calculated as the fluorescence intensity relative to reactions treated with compound diluents, following subtraction of background fluorescence from each sample.

2.6.9. Transcriptome analysis

Overnight cultures of S. aureus SH1000 were diluted 1:100 into 27 ml of MHB and incubated at 37oC with aeration and shaking to an OD600 of 0.2 units. At this point 3 ml of AgNO3 was added at a concentration that would inhibit growth of cultures by 25% (relative to a drug-free controls, into which 3 ml of sterile H2O was added) over a 40 min period (Freiberg, et al., 2005). Following incubation, 10 ml volumes were transferred into 50 ml falcon tubes containing 20 ml of RNA protect bacterial reagent (Qiagen, Manchester, UK), vortexed for 10 sec, and then incubated without shaking at room temperature for 10 min. Following centrifugation of samples (at 5000 x g) and removal of supernatant, cell pellets were washed twice in TE buffer (10 mM tris (hydroxymethyl) aminomethane, 1 mM ethelynediaminetetra-acetic acid [pH 8.0]),

re-suspended in TE buffer containing 200 g RNAse-free lysostaphin/ml, and then incubated at 37oC for 90 min with gentle mixing every 15 min. RNAse-free Proteinase K was then added to a final concentration of 40 g/ml and the cell suspension incubated at room temperature for a further 10 min. Total RNA was extracted from cell suspensions using an RNAeasy midi kit (Qiagen) according to manufacturer‘s instructions, and eluted in 500 l of nuclease-free H2O.

Determination of RNA quality, synthesis and hybridisation of cDNA to microarrays and subsequent analysis (i.e. fluorescence measurements and normalisation) was performed by Roche Nimblegen (Madison, Wisconsin, USA). Microarray probe sequences were derived from the chromosomal DNA sequence of S. aureus 8325 and fluorescence values were normalised using quantile normalisation.

Results from microarray experiments were subsequently analysed using ArrayStar 4 software (DNAstar, Madison, Wisconsin, USA) and a ≥2-fold difference in gene expression between control and test samples (at a 99% confidence limit) was considered significant.

2.6.10. Screening of a near-saturation transposon library of S. aureus

A library of S. aureus SH1000 comprising ~20,500 transposants was screened for Ag+ hypersusceptibility according to a method described previously (Blake & O'Neill, 2013). The concentration of AgNO3 selected for screening transposants was determined as the concentration that inhibited the growth of 1-2 library strains per 96-well plate, without inhibiting growth of the parental strain under identical conditions.