Study of Antibacterial Activity by Capillary Electrophoresis Using

Article pubs.acs.org/ac

Study of Antibacterial Activity by Capillary Electrophoresis Using Multiple UV Detection Points Farid Oukacine,†,‡ Bernard Romestand,§ David M. Goodall,⊥ Gladys Massiera,# Laurent Garrelly,‡ and Hervé Cottet*,† †

Institut des Biomolécules Max Mousseron (IBMM, UMR 5247 CNRS-Université de Montpellier1- Université de Montpellier 2), Place Eugène Bataillon, case courrier 1706, 34095 Montpellier Cedex 5, France ‡ COLCOM, Cap Alpha, Avenue de l’Europe, Clapiers 34940 Montpellier, France § Laboratoire Ecosystèmes Lagunaires, UMR 5119, Université de Montpellier 2, 34095 Montpellier, France ⊥ Paraytec Ltd, York House, Outgang Lane, Osbaldwick, York YO19 5UP, United Kingdom # Laboratoire Charles Coulomb, UMR 5521, CNRS-Université de Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France ABSTRACT: A new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gramnegative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm ×100 μm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-L-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-L-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications. total internal reflection microscopy (TIRM),14 and optical tweezers.12 During the past decade, there has been considerable interest in bacteria analysis by capillary electrophoresis (CE). Hjertén et al. were the first to introduce the idea of microbial analysis by CE.15 They showed that tobacco mosaic virus and Lactobacillus casei migrated through a capillary under an electric field. Subsequently, research efforts have been carried out to develop new CE methods for bacteria analysis. However, due to the physiological heterogeneity of microbial population,16 bacteria analysis by CE has proved to be more difficult than for small molecules. It is not possible to use capillary zone electrophoresis (CZE) for reproducible and quantitative analysis of bacteria.17 In order to improve the reproducibility, selectivity and sensitivity of microbial analysis, a number of different approaches have been reported.17−21 Different microbial assay applications based on CE techniques have been developed. Among them, we can mention viability determination of bacteria,22a quick sterility test,23 microbial contamination in real

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he excessive use of antimicrobials both in humans and animal husbandry has led to emergence of bacteria strains which are resistant to a range of antimicrobials, including firstchoice agents for the treatment of humans.1 Thus, the emergence of resistance to antibacterials requires the search for new antiinfective compounds2−5 and rapid identification of their antibacterial efficiencies. Established methods for assessment of the efficiency of compounds are the broth microdilution method, the disk diffusion method,6−8 the BACTEC, and the microplate alamar blue assay (MABA).9,10 The inability of these tests to distinguish between cytotoxic and cytostatic effects requires an additional viability enumeration of the drugtreated cell population. This is traditionally assessed by counting the colony forming units (CFU) arising from surviving bacterial cells.11 Unfortunately, these procedures have the disadvantage of being time-consuming. Furthermore, there is an increasing need for efficient methods that allow estimation of the magnitude of the binding of molecules to microorganisms. Indeed, the adhesion of microorganisms to the surface of host tissue is often a first step in pathogenesis and is plausible target for new antiinfective agents.12 Nonelectrophoretic methods used to determine molecule−microbe specific binding interactions include atomic force microscopy (AFM),13 © 2012 American Chemical Society

Received: January 1, 2012 Accepted: February 29, 2012 Published: March 20, 2012 3302

dx.doi.org/10.1021/ac300004t | Anal. Chem. 2012, 84, 3302−3310

Analytical Chemistry

Article

biological samples,24 and quantification of bacteria in natural waters.25 However, only a few reports have focused on using CE to study the interaction of bacteria with molecules. Armstrong et al. used affinity capillary electrophoresis (ACE) to evaluate the binding constant of Bifidobacterium infantis with Lucifer yellow and vancomycin.26 By adding B. infantis at different concentrations in the running buffer and by electrophoretic mobility measurements of these molecules, it is possible to evaluate the binding constant. Torimura et al. evaluated the redox enzymatic activity of microorganisms by CZE using an appropriate substrate and an exogenous electron acceptor.27 Hoerr et al. reported a new method for the quantification of the effect of an antibiotic by laser-induced fluorescence capillary electrophoresis (LIF-CE). Before injection into the capillary, the bacteria were incubated in a drug solution during 18 h and were then analyzed by CE using LIVE/DEAD Bac light viability kit composed of the two fluorescent nucleic acid stains, SYTO9 and propidium iodide.28 The aim of this work was to investigate the possibility to use CE for the evaluation of antibacterial activity. For that, a UV detector imaging three windows was implemented to monitor the electrophoretic profile of bacteria before, during, and after the meeting in the capillary with a potentially antimicrobial cationic compound. This new analytical methodology was used to study the effect of the second generation of dendrigraft polyL-lysines (DGL) on Micrococcus luteus and Erwinia carotovora.

removed carefully, and the pellet was resuspended in 8 mL of CE terminating electrolyte (13.6 mM Tris, 150 mM boric acid) by vortexing for 1 min. The bacteria suspension was centrifuged again for 5 min. This washing process was repeated twice. Eight milliliters of CE-terminating electrolyte was added to the washed bacteria cells, and the solution was vortexed until the pellets were resuspended completely. Subsequently, the bacterial suspensions were stored at 4 °C. Cell concentrations were measured by turbidity at 600 nm (model: Uvikon 860, Kontron Instruments, Zürich, Switzerland). It was assumed that the turbidity for all bacteria strains are the same (1 AU recorded at 600 nm using 1 cm path length cell corresponds to a cell concentration of 6 × 108 cell mL−1).29 Before use, filtration of the buffer is required to remove any possible bacterial contamination or particles from the buffer. The filtration was performed using MF-Millipore filters with 0.45 μm pore size (Millipore, Molsheim, France). Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Antibacterial assays were performed with Gram-positive (M. luteus) and with Gram-negative bacteria (E. carotovora). MIC and MBC were determined for benzylamine, DGL (G2), and carboxy fluorescein-labeled G2. A broth dilution method was used to determine the MIC and the MBC. Briefly, about 30 °C overnight cultures were inoculated in poor broth nutrient medium (1% bactotryptone and 0.5% NaCl (w/v), pH 7.5).30 The optical density (OD) of the inoculum was measured at 600 nm and diluted about 1000 times to reach a final optical density of 0.001. Stock solutions of benzylamine, G2, and carboxy fluorescein-labeled G2 were prepared in sterile distilled water and then diluted at various concentrations. Ten microliter aliquots from each dilution were incubated in microtiter plates (96 wells) with 100 μL of a suspension of midlogarithmic phase culture of bacteria at a starting optical density of OD600 nm = 0.001. The microtiter plates containing the cultures were grown in the dark at 30 °C during 24 h under slight shaking. The MIC was defined as the lowest concentration of the antimicrobial compound for which the microorganisms do not demonstrate visible growth. Thus, growth inhibition was controlled by measurement of the optical density at 600 nm after 24 h incubation. Cell suspensions (100 μL) from the microtiter plates showing no growth were subcultured on poor broth agar plates to determine if the inhibition was reversible or permanent. MBC was determined as the highest dilution (lowest concentration) for which no growth occurred on the agar plates.31 The quantitative MIC and MBC results are gathered in Table 1. The same protocol, with slight modifications, was used for the determination of the MBC of G2 at high salt concentration (154 mM NaCl) or low salt concentration (15.4 mM NaCl) and in a 10 fold diluted poor

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EXPERIMENTAL SECTION Chemicals. Boric acid 99.999% (H3BO3), Tris 99.9% (HOCH2)3CNH2, hydrochloric acid 99.999%, benzoic acid (C7H6O2), benzylamine 99% (C6H5CH2NH2), 2-propanol (C3H8O), and hydroxypropyl cellulose (Mw 105 g mol−1) were purchased from Aldrich (Steinheim, Germany). The second generation of dendrigraft poly-L-lysines (G2) and carboxy fluorescein-labeled G2 were supplied by COLCOM (Montpellier, France) and used without further purification. Deionized water was further purified with a Milli-Q system from Millipore (Molsheim, France). Nutrient broth medium was purchased from Difco Laboratories (Franklin Lakes, NJ). Luria−Bertani (LB) agar plates and the bacteria Micrococcus luteus (ATCC 4698) and Erwinia carotovora (from IBMC Strasbourg collection) were kindly donated by Lagoon Ecosystems laboratory (University of Montpellier 2). Bacterial Growth Conditions and Sample Preparation. The different bacterial species were maintained under strictly controlled growth conditions: the starting culture colonies of the bacteria (M. luteus and E. carotovora) were transferred from LB agar plates into Erlenmeyer flasks containing 8 mL of LB liquid medium. The flasks were incubated for 13 h at 30 °C with constant agitation (120 rpm) on a rotary platform shaker for good aeration. Then, 80 μL of a bacteria suspension were transferred into Erlenmeyer flasks containing 8 mL of LB liquid medium. The flasks were incubated another time at 30 °C with constant agitation (120 rpm) until the turbidity at 600 nm of the bacteria suspension in a 1 cm path length cell reached 100− 200 mAU. Such values of turbidity are characteristic of bacteria in an exponential growth phase. This allows limiting the substantial cell-to-cell differences in physiological parameters such as growth rate and resistance to stress. The fresh liquid cultures were prepared daily. To separate the bacterial culture from the medium, the suspension in the flask was centrifuged (model: SIGMA 3K12, Osterode, Germany) at 6000 rpm for 5 min to get the bacteria in pellet form. The supernatant was

Table 1. Antimicrobial Activity Expressed in Terms of MICa and MBCa in mg L−1 for Benzylamine, G2, and Carboxy Fluorescein-Labeled G2 on M. luteus and E. carotovora benzylamine M. luteus E. carotovora

carboxy fluoresceinlabeled G2

G2

MIC

MBC

MIC

MBC

MIC

MBC

>500 >500

>500 >500

6.7 >500

13.4 >500

1.7 >500

3.4 >500

a

MIC: minimum inhibitory concentration. MBC: minimum bactericidal concentration.

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RESULTS AND DISCUSSION Implementation of the Methodology Used To Study the Antibacterial Activity of Cationic Molecules on Bacteria (Gram+ and Gram−). Different approaches that allow focusing bacteria in capillary electrophoresis have been reported. These approaches include isotachophoretic focusing (ITP),17 large volume sample stacking with polarity switching,21 capillary isoelectric focusing,19,20 and pH-induced stacking and normal stacking mode.18 For implementation of the method used in this work, the mobilization of bacteria in the capillary by anionic ITP appeared to be the most appropriate. Indeed, the pH used in anionic ITP of bacteria17 is very close to the physiological pH, and this focusing mode is usable in a neutral coated capillary for which there are no electrostatic interactions between dendrigraft poly-L-lysines and the capillary wall.37 The electrolytes used for the ITP focusing were Tris/boric acid at 13.6/150 mM as terminating electrolyte (TE) and Tris/boric acid/HCl at 4.5/50/3.31 mM as leading electrolyte (LE). In this ITP system, boric acid acts as terminating ion and chloride acts as leading ion.17 Figure 1 gives a schematic outline of the methodology used in this work. UV area imaging detector (ActiPix D100,

broth nutrient medium. In that protocol, the inoculum with OD600 nm = 0.1 was diluted to reach final optical density of OD600 nm = 0.001 in three different media (NaCl at 154 mM, NaCl at 15.4 mM, and 10 fold diluted poor broth nutrient medium). Capillary Coating. The hydroxypropyl cellulose (HPC)coated capillaries were prepared according to a literature protocol.32,33 HPC powder (Mw 105 g mol−1) was dissolved at room temperature in water to a final concentration of 5% (w/ w). The polymer solutions were left overnight to eliminate bubbles. The 1 m length capillary columns were filled with the polymer solution using a syringe pump (KDS100, Holliston, MA) for 30 min, and the excess of polymer solution was removed using N2 gas at 0.4 bar. The HPC polymer layer was immobilized by heating the capillary in a GC oven (GC-14A, Shimadzu, France) at 60 °C for 10 min and using a linear ramp from 60 to 140 °C at 5 °C/min, and finally 140 °C for 20 min, keeping N2 pressure at 0.4 bar. Before use, the coated capillaries were rinsed with water for 10 min. At the end of each day, the HPC-coated capillary was flushed with water for 10 min and with air for 5 min. Capillary Electrophoresis. CE experiments were carried out with a 3D-CE instrument (Agilent Technologies System, Waldbronn, Germany) equipped with a diode array detector. Separation capillaries prepared from bare silica tubing were purchased from Composite Metal Services (Shipley, UK). The temperature of the capillary cassette was maintained constant at 25 °C. Leading electrolyte (LE) was composed of 4.5 mM Tris + 50 mM boric acid + 3.31 mM HCl. Terminating electrolyte (TE) was of 13.6 mM Tris + 150 mM boric acid. The pH values of leading and terminating electrolytes are respectively 7.28 and 7.94. Between each run, the capillary was washed with LE for 10 min. Immediately prior to the injection, the bacteria samples were vortexed during 1.5 min to avoid bacteria sedimentation in the vial. Multiple Window UV Detection. UV detection at three independent windows in a looped capillary was carried out with an ActiPix D100 UV area imaging detector (Paraytec, York, UK).34,35 The light source was a pulsed xenon lamp, and the selected wavelengths were 280 and 214 nm using filters with 20 nm bandpass (full width at half-maximum). The sample rate used for acquisition was 20 Hz, and the detector constant time was 0.1 s. Computer Simulation. For computer simulations, the freeware program Simul 5.0 by Gaš et al.36 was used (available at http://natur.cuni.cz/gas). The simulation was performed with 1000 grid points along a 25 mm long separation space for a 0.5 mm long inlet zone, 3.5 mm long sample zone, and 21 mm long outlet zone. Simulation was performed at a constant voltage of 250 V and without electroosmotic flow. The inner diameter of the capillary was 50 μm, and the optimization interval was 100 steps. Microscopy. Microscopy observations were performed using an inverted Olympus microscope (IX71) equipped with an oil-immersion objective (100× N.A. 1.3) and an additional 1.6× lens. For epifluorescence observations, excitation is provided by a 100 W mercury lamp and a 460−490 nm band-pass filter. The bacteria solutions are observed in a closed microscope chamber (two glass slides spaced by vacuum grease). Bright field and fluorescence images are detected using a Hamamatsu camera (Orca-R2). The samples are observed approximately 10 min after preparation to let the bacteria sediment at the bottom of the chamber.

Figure 1. Schematic representation of the methodology used to study the antibacterial activity of cationic compounds. The bacterial zone is injected at the cathode while the cationic compound is injected at the anode. Three independent UV detection points allow monitoring of the bacterial zone before, during, and after the meeting with the cationic compound. See Experimental Section for details on the compositions of leading and terminating electrolytes (LE and TE, respectively).

Paraytec) was used with a 95 cm × 100 μm i.d. HPC-coated capillary having two loops and three independent detection windows. The spacing between neighboring detection windows was 15 cm. As described in Figure 1, the distance between the cathodic (inlet) end of the capillary to the first detection window is 27 cm, and the distance between the anodic (outlet) end of the capillary and the third (last) detection window is 38 cm. The bacteria sample was injected at the cathodic end of the capillary, and the cationic molecule was injected at the anodic end of the capillary. This allows monitoring the electrophoretic profile of the bacteria plug before, during, and after the meeting with the cationic compound. In this methodology, the bacteria plug moves under anionic ITP conditions, allowing the bacteria zone to keep focused, while the cationic compound is displaced under nearly zone conditions because LE and TE are both based on Tris cation. In CE the injected volume does not usually exceed 1% of the total capillary volume. For this injected volume, band broadening of the sample peak,38 during the electrophoretic process, leads to dilution of the sample zone. Therefore, classical hydrodynamic injection would not allow a control of the concentration in the zone of the cationic molecule before and during its meeting with the bacteria plug. In the case of large sample volume, the concentration in cationic molecule at the plateau remains constant. 3304



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Figure 2A (trace 2). Similar results were obtained with a critical concentration of about 2.2 mM in benzylamine above which the system renormalizes the benzylamine concentration. Figure 2B displays the concentration distributions for Tris and benzylamine in the capillary at three different times of the CZE process. At t0, just after the injection, two distinct zones are present in the capillary: zone 1 corresponding to the Tris/ borate BGE and zone 2 corresponding to the injected zone containing benzylamine in BGE. For each zone, the Kohlrausch regulating function (KRF) has values (w1 and w2, respectively) which are given by eq 1:39

Figure 2A displays the UV absorbance at the plateau of a benzylamine band in the case of a pressure-driven front (trace

wi = ∑

Figure 2. Comparison of the front heights obtained for pressuredriven flow and in CZE mode for benzylamine samples at different concentrations (A), and computer simulation showing the concentration distributions of benzylamine and Tris in the capillary in CZE mode (B). (A) Experimental conditions: HPC-coated capillary 80 cm (27 cm to the detection window) × 100 μm i.d. Electrolyte: LE, Tris 4.5 mM + boric acid 50 mM + HCl 3.31 mM. Sample: benzylamine diluted in LE (concentration as indicated on the graph). Mobilization pressure (trace 1) at 30 mbar. Applied voltage: 30 kV (trace 2) after sample injection at 50 mbar for 60 s. Detection at 214 nm (2 nm bandwidth). (B) Simulation conditions: times courses, t0 = 0 s, t1 = 17.60 s, t2 = 40.72 s. The simulated separation was performed along a 25 mm long separation space for a 0.5 mm long inlet zone (LE), 3.5 mm long sample zone, and 21 mm long outlet zone (LE). Sample: 10 mM benzylamine in LE. Other conditions: see computer simulation.

Cj

j |mj|

(1)

where Cj and mj refer to the concentrations and ionic mobilities at infinite dilution of all species j present in zone i. Immediately after the voltage is applied, a new zone containing benzylamine appears (zone 3) such that w3= w1. As depicted in Figure 2B, the boundary between zone 2 and zone 3 remains stationary during the whole electrophoretic process. The concentration of benzylamine in zone 3 is regulated by the system such that w3 is maintained equal to w1. As a consequence, the benzylamine concentration in zone 3 depends on the TrisH+ concentration in zone 1 and the ratio of the effective mobilities of Tris and benzylamine. For a low injected concentration of benzylamine (≤2.2 mM), w1 and w2 are approximately the same and the regulation of benzylamine concentration by the system is negligible. From these preliminary results, it can be concluded that, using the experimental conditions described, it is possible to study the possible antibacterial activities of a cationic band (typically representing 16% of the total capillary volume) on a bacteria plug, provided that the injected concentration is lower than ∼2 mM. Two sequential steps were used to implement the antibacterial assay by capillary ITP. In the first step, the capillary was washed by LE, and the tested antibacterial cationic molecule (diluted in LE) was hydrodynamically injected from the outlet end of the capillary (anodic end) as a broad band.

1) versus CZE mode at different injected concentrations in benzylamine. In CZE mode, the injected volumes of benzylamine correspond to 16% of the total capillary volume. CZE was performed in LE. It appears from Figure 2A that below a critical benzylamine concentration of ∼2.2 mM, there is no dilution of the sample in the capillary during the CZE process, because the UV absorbance at the plateau was similar to that obtained for a pressure-driven flow front. Above 2.2 mM benzylamine, the plateau height tends to level off because of a renormalization of the benzylamine concentration by the electrophoretic system. A computer simulation (Simul 5.0)36 was carried out using the same experimental conditions as in

Figure 3. Electropherograms obtained for the meeting beetween benzylamine (cationic compound) and benzoate (A), M. luteus (B), or E. carotovora (C) in a single capillary with three independent windows. Experimental conditions: HPC-coated capillary 95 cm with two loops and three detection windows (27.5 cm to the 1st (trace 1), 42.5 cm to the 2nd (trace 2), and 57.5 cm to the 3rd detection window (trace 3) from the inlet end of the capillary) × 100 μm i.d. 1st step: 10 min washing at 1000 mbar with LE. Hydrodynamic injection at the outlet end of the capillary of the cationic zone (benzylamine at 0.123 g L−1 in LE) at 50 mbar for 80 s. Mobilization of the benzylamine zone in zone mode (LE): −30 kV for 4.5 min. 2nd step: Hydrodynamic injection of the anionic zone at the inlet end of the capillary: 50 mbar for 1.5 s (A), 50 mbar for 10 s (B, C). Samples: benzoic acid at 0.617 g L−1 in LE (A), M. luteus at 2.03 × 108 cells mL−1 in LE (B), E. carotovora at 0.72 × 108 cells mL−1 in LE (C). After injection, ITP is carried out at a constant current of 3 μA with TE at the inlet end and LE at the outlet end. See Experimental Section for details on the composition of leading (LE) and terminating (TE) electrolytes. 3305



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Figure 4. Electropherograms obtained for the meeting between G2 (cationic compound) and benzoate (A), M. luteus (B), or E. carotovora (C) in a single capillary with three independent windows. Experimental conditions: (1st step) Hydrodynamic injection at the outlet end of the capillary of the cationic zone (G2 at 0.498 g L−1 in LE) at 50 mbar for 80 s. Mobilization of the G2 zone in zone mode (LE): −30 kV for 2 min. (2nd step) Samples: benzoic acid at 0.617 g L−1 in LE (A), M. luteus at 1.85 × 108 cells mL−1 in LE (B), E. carotovora at 0.72 × 108 cells mL−1 in LE (C). Other conditions are as described in Figure 3.

the terminating compartments. In trace 3, benzylamine is in the leading compartment where the local electric field is low (∼85 V cm−1) vs 330 V cm−1 in the terminating compartment (trace 1). Thus, the velocity of benzylamine at the third detection window is much lower than at the first detection window. As a consequence, the peak area of benzylamine is not constant before and after the meeting with the anionic plug even though the quantity of benzylamine is conserved. By contrast, because of the anionic ITP process, the peak area of benzoate remains constant (∼3200 mAU s) at all three detection windows. Comparison of the electrophoretic profiles of benzylamine obtained before and after the meeting with benzoate (trace 3 vs trace 1 in Figure 3A) does not show any significant change in band shape that could reflect any interaction between oppositely charged compounds. Figure 3B and 3C displays results of the meetings between benzylamine and M. luteus (Gram-positive bacteria) and E. carvotovora (Gram-negative bacteria), respectively. For each experiment, electropherograms are presented at two different wavelengths, 214 nm and at 280 nm, because the signal for benzylamine was not significant at 280 nm whereas the bacteria were detected at both wavelengths. The two wavelengths were recorded on two independent runs because the detector does not allow simultaneous acquisition of data at different wavelengths. The main information from Figure 3B and 3C is that the bacteria plug was not affected by the meeting with the benzylamine zone (trace 1 vs trace 3), demonstrating the absence of fast bacteriolytic activity of benzylamine against bacteria. These results are in agreement with the minimal bactericidal concentration (MBC) values obtained by traditional tests used in microbiology (see Table 1) because the MBC values for benzylamine on M. luteus and E. carotovora are very high (>500 mg L−1 corresponding to 4.7 mM). Furthermore, the MBC of benzylamine (pKa ∼ 11) is determined by the proportion of the free base form, ∼10−4× the level of the protonated benzylamine form at the pH of the experiment, pH 7. Regarding the benzylamine electrophoretic profile, there is no significant change in its shape before (trace 3, 214 nm) and after (trace 1, 214 nm) the meeting with M. luteus or E. carotovora, except for the band height being higher in trace 3 than in trace 1 due to a renormalization of the benzylamine concentration in its zone according to the KRF. Because the TrisH+ concentration is higher in the leading

Using LE as BGE in both inlet and outlet vials and operating in CZE mode, −30 kV voltage was next applied for a given time (tcat+,cze), which was selected dependent on the electrophoretic mobility of the cationic molecule. In the second step, the bacteria sample diluted in TE was injected at the inlet (cathodic) end of the capillary. Using LE in the outlet vial and TE in the inlet vial, anionic ITP was then performed at a constant current (3 μA). tcat+,cze was calculated so that the time remaining for the cationic molecule to reach the second detection window in ITP mode under an electrical current of 3 μA, is equal to the time required for the bacteria plug to reach the same detector. For more detail on the calculation of tcat+,cze, refer to Appendix. Effect of Benzylamine (blank assay) on Benzoate, M. luteus, and E. carotovora Using Capillary ITP and Multiple UV Detection Windows. Figure 3A displays the electropherograms obtained at three independent UV detection windows (traces 1 to 3) for the meeting between a small anionic molecule (benzoate) and cationic molecule (benzylamine) according to the methodology previously described. Benzoate is detected at 214 nm before the meeting with benzylamine (cationic) in trace 1 and after the meeting with benzylamine in trace 3. Conversely, benzylamine is detected before (trace 3) and after (trace 1) the meeting with benzoate. The arrows on the graph are guides for the eye and indicate the direction of the evolution of the electrophoretic profiles of the molecules during their displacements in the capillary. The small peak marked with an asterisk in trace 3 corresponds to impurities coming from the buffer and focused at the interface between leading and terminating electrolytes (not uncommon in ITP, especially in the UV range of 193−210 nm).40,41 This small peak appears also for the ITP analysis of bacteria (see Figure 3B and 3C). The space between this small peak and the benzoate peak is attributable to the presence of hydrogen carbonate/carbonate which plays the role of a spacer in anionic ITP. Indeed, most buffer solutions used in anionic ITP contain dissolved carbon dioxide because of its high solubility in aqueous solutions,42 depending on the pH used and the ionic strength of the buffer. Note that the bandwidth of benzylamine is much higher in trace 3 (detection window no. 3) than in trace 1 (detection window no. 1). In trace 3, the signal of benzylamine is obtained before its meeting with the interface, delimiting the leading and 3306



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Figure 5. Microscopy pictures of M. luteus (A) and E. carotovora (B) showing the effect of G2 on the bacteria. Experimental conditions: M. luteus at 1.50 × 108 cells mL−1 (A-1); M. luteus at 1.50 × 108 cells mL−1 + G2 at 501 mg L−1(A-2); E. carotovora at 1.51 × 108 cells mL−1 (B-1); E. carotovora at 1.51 × 108 cells mL−1 + G2 at 501 mg L−1(B-2); E. carotovora at 1.51 × 108 cells mL−1 + carboxy fluorescein-labeled G2 at 25 mg L−1 (B-3). Scale bar is 15 μm. Pictures A1-2 and B1-2 were obtained using bright field microscopy. Figure B-3 was obtained using epifluorescence to image carboxy fluorescein-labeled G2.

Figure 6. Electrophoretic profiles of M. luteus (A) and E. carotovora (B) in anionic isotachophoretic mode (ITP) or in zone mode (CZE) before (traces 1 to 3) and after (traces 4 to 6) incubation of the bacteria in the presence of G2. Experimental conditions: HPC-coated capillary 33.5 cm (25 cm to the detector) × 100 μm i.d. Samples: M. luteus at 7.98 × 108 cells mL−1 (A), E. carotovora at 7.02 × 108 cells mL−1 (B), both in TE (traces 1 to 3) or in TE containing 0.498 g L−1 G2 (traces 4 to 6). Hydrodynamic injection: 17 mbar, 6 s. ITP mode: LE in the capillary and in the outlet vial, TE in the inlet vial. Current: 3 μA. CZE mode: ±15 kV as indicated on the graph. Electrolytes: TE (2, 3), TE containing 0.498 g L−1 G2 (5, 6).

Figure 4B the signal of M. luteus completely disappeared after contact with the G2 band (see trace 1 vs trace 3 at both 214 and 280 nm). At the same time, the electrophoretic profile of G2 was not modified after the meeting with the microbial band. We can conclude from this assay that M. luteus is lysed after contact with G2, in good agreement with the low MBC (13.4 mg L−1 corresponding to 1.6 μM, see Table 1). Lysis of M. luteus in the capillary after contact with the G2 band is also supported by microscopy imaging. Figure 5A-1 and 5A-2 displays microscopy images of M. luteus suspensions without and with G2 (528 mg L−1), respectively. In the absence of G2 in the medium, M. luteus appears as tetrads and as irregular clusters of tetrads. The presence of G2 followed with 2 min vortexing leads to lysis with complete destruction of bacteria (Figure 5A-2). It should be noticed that the presence of G2 alone does not lyse M. luteus. An additional constraint such as the physical shear of the vortex is required. The impact of the vortexing on the complete destruction of M. luteus could suggest that G2 triggers the formation of bacteria clusters that are then more sensitive to shear forces. This phenomenon has been also reported by Levashov et al.47 for the lysis of Gram-negative bacteria by lysozyme. In the case of the lysis of M. luteus in CE after the meeting with the G2 plug, we assume that the additional constraint is the electric field applied during the electrophoretic process.

compartment than in the terminating one, the benzylamine concentration is higher before crossing (trace 3) the ITP interface than after (trace 1) as set by the KRF. Trace 2 clearly shows this renormalization in the benzylamine concentration at the ITP interface. Antibacterial Effect of a Second Generation of Dendrigraft Poly-L-lysines on M. luteus and E. carotovora Using Capillary ITP and Multiple UV Detection. Recently, our group described a new synthetic route to dendrigraft polyL-lysines (DGL) by polymerization of N-carboxyanhydrides of amino acids (NCA) in water. 43 DGLs are dendritic polypeptides that are biocompatible, are nonimmunogenic44 and have low toxicity.45 While five successive generations were routinely synthesized,43 we mainly focused in this study on the second generation (G2). Briefly, G2 is a branched poly-L-lysine with a number-average degree of polymerization of 48 lysine residues (9 α-amine groups and 40 ε-amine groups). The polydispersity index of G2 is 1.38, and the hydrodynamic radius measured at pH 7 in a phosphate buffer (0.306 M ionic strength) is 1.40 nm. More details on the structure of DGLs are given elsewhere.43,46 Figure 4 displays the antibacterial assay between G2 and benzoate (blank, 4A), M. luteus (4B), or E. carotovora (4C) using the present methodology. Figure 4A shows that there is no specific interaction between G2 and benzoate. By contrast, in 3307



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Figure 4C displays the antibacterial assay between G2 and E. carotovora. The signal of E. carotovora disappears after the meeting in the capillary with G2 (trace 3 vs trace 1 at 280 nm). However, a new bacteria peak appears in trace 1 at around 15 min (trace 1 at 280 nm). The electrophoretic profile of G2 observed at 214 nm (trace 1) is also modified after contact with the bacteria plug because a small hump appears at ∼15 min. These observations can be explained by a change in the bacteria charge before (negative charge) and after (positive charge) the meeting with the G2 zone due to G2 adsorption onto the outer membrane of the bacteria. Indeed, the bacteria plug is detected a first time by detector #1 (resulting in a sharp peak at ∼7.5 min in trace 1 of Figure 4C). In a second step, the bacteria plug meets the G2 (cationic molecule) in the capillary at ∼11 min. Because of G2 adsorption onto the outer membrane of the bacteria, the bacteria become positively charged and the direction of mobilization changes. Consequently, the bacteria plug is detected a second time together with the G2 band by detector #1 (resulting in a hump at ∼15 min in trace 1 of Figure 4C). To confirm the change in bacteria charge due to G2 adsorption, additional experiments were carried out using a short capillary with only one detection point. Figure 6 displays the electrophoretic profiles of M. luteus (6A) and E. carotovora (6B) in anionic isotachophoretic mode (ITP) or in zone mode (CZE) in the absence or in the presence of G2. In the absence of G2, Figure 6A-1 and 6B-1 shows the expected focused bands for living bacteria in anionic ITP mode. For the same samples, Figure 6A-2 and 6B-2 displays the characteristic electrophoretic profiles of bacteria in zone mode showing a forest of small peaks in negative polarity (−15 kV). As expected, in positive polarity, Figure 6A-3 and 6B-3 demonstrate that there is no bacteria signal because both bacteria strains are negatively charged at pH 7. In the presence of 498 mg L−1 of G2 in the bacteria samples, Figure 6A (traces 4 to 6) demonstrates the full lysis of M. luteus because flat traces were obtained in all the electrophoretic profiles in ITP and CZE modes. By contrast, in the case of E. carotovora, the presence of G2 extinguished the focused band in the ITP signal (trace 4) and the CZE trace in negative polarity (trace 5) while a multitude of small peaks could be detected in CZE with positive polarity (trace 6), demonstrating the change in charge polarity on the outer membrane of E. carotovora due to G2 adsorption. Figure 5B displays the microscopy imaging of E. carotovora suspensions in the absence (B-1) and in the presence (B-2) of G2, respectively. In both images, E. carotovora appears as a rod-shaped bacteria, and it is not possible to visualize, from the microscopy imaging, adsorption of G2 at the surface of E. carotovora. The unique way to visualize adsorption of G2 onto the surface of bacteria was to use a fluorescent derivative of G2 combined with imaging by fluorescence microscopy. Figure 5B-3 clearly shows that carboxy fluorescein-labeled G2 was detected at the surface of the bacteria, confirming the results obtained by the CE assay. There are two advantages of CE versus microscopy imaging for the investigation of interaction between bacteria and small molecules: (i) fluorescent labeling is not required and (ii) the possible adsorption due to nonspecific interaction of the fluorescent dye onto the bacteria is discarded in CE with no possibility of leading to false-positive tests. We were somewhat surprised by the fact that despite the very strong interaction existing between G2 and E. carotovora, there is no bactericidal activity of G2 against E. carotovora. Indeed, it is well-known that evidence for interaction with the surface of

the microorganism is often a first step in the search of new antiinfective agents.12 The differences between CE conditions used in this work and the microbiological conditions used for the determination of the MBC are mainly the ionic strength and the presence of several proteins and other nutriments in the culture broth. MBC values for G2 on E. carotovora were measured at high salt concentration (154 mM NaCl), low salt concentration (15.4 mM NaCl), and in a 10-fold diluted poor broth nutrient medium. The MBC values obtained were respectively >500 mg L−1, < 3.2 mg L−1, and 52 mg L−1. It should be noticed that there are no growth inhibitions for all the control samples, showing that the bactericidal activity of G2 at low salt concentration is not due to osmolysis. Similar behavior was reported for some divalent dendrimers tethered with two copies of an octapeptide (RLYRKVYG). Under low salt conditions (10 mM phosphate buffer without NaCl), the divalent dendrimeric octapeptide showed high activity against E. coli (MIC < 1 μM). However, under high salt conditions (10 mM phosphate buffer with 100 mM NaCl), the divalent dendrimeric octapeptide did not retain its antimicrobial activity against this bacterial strain (MIC ∼ 30 μM).48 We assume that the mechanism of action of G2 on M. luteus is essentially based on electrostatic interactions with the outer membrane of the bacteria. Thus, at high ionic strength, the surface charges of G2 are screened by a cloud of counterions decreasing the strength of interaction with the bacteria. However, the ionic strength may also have an impact on the bacteria aggregation and/or on the configuration of the cationic dendrimers itself that can adopt a more compact form. In conclusion, under low-salt conditions, G2 demonstrates very high activity against E. carotovora (Gram-negative bacteria). However, this activity is completely lost under high salt conditions and/or in the presence of nondiluted nutrient medium.

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CONCLUSION A new methodology based on anionic ITP and multiple UV detection points has been developed for evaluation of antibacterial activity and molecule−microbe interactions. This methodology was found very useful and effective to study the fast bacteriolytic activity of cationic compounds or to study the adsorption of cationic molecules onto bacteria. The results obtained showed a fast bacteriolytic activity of the second generation of DGL (G2) against Micrococcus luteus (Grampositive bacteria). The bactericidal activity of G2 against E. carotovora (Gram-negative bacteria), under low-salt conditions, has been also highlighted, but this activity is completely lost under high salt conditions. Because DGLs are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications. Chemical modifications of the DGL surface is under investigation for improving the bactericidal properties of DGL against Gram-negative bacteria.

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APPENDIX The time tbacteria,itp required for the bacteria to reach the detection window #2 under ITP conditions is given by: t bacteria,itp =

d |μCl−,LE|ELE,itp

(1)

where d is the distance from the cathodic end of the capillary to the meeting point (42 cm), μCl−,LE is the effective mobility of 3308



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(2) Biedenbach, D. J.; Jones, R. N. J. Clin. Microbiol. 1997, 35, 3198− 3202. (3) Cormican, M. G.; Jones, R. N. Drugs 1996, 51, 6−12. (4) Fung, H. B.; Kirschenbaum, H. L.; Ojofeitimi, B. O. Clin. Ther. 2001, 23, 356−391. (5) Talbot, G. H.; Bradley, J.; Edwards, J. E.; Gilbert, D.; Scheld, M.; Bartlett, J. G. Clin. Infect. Dis. 2006, 42, 657−668. (6) National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically, 4th ed.; Approved standard M7-A4; National Committee for Clinical Laboratory Standards, Villanova, PA, 1997. (7) National Committee for Clinical Laboratory Standards. Development of in Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved guideline M23-A; National Committee for Clinical Laboratory Standards, Villanova, PA, 1994. (8) National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved standard M2-A6; National Committee for Clinical Laboratory Standards, Villanova, PA, 1997. (9) Protopopova, M.; Hanrahan, C.; Nikonenko, B.; Samala, R.; Chen, P.; Gearhart, J.; Einck, L.; Nacy, C. A. J. Antimicrob. Chemother. 2005, 56, 968−974. (10) Burkhardt, F. Mikrobiologische Diagnostik, 1st ed.; Thieme: Stuttgart, Germany, 1992. (11) Bast, E. Mikrobiologische Methoden, 2nd ed.; Spektrum: Heidelberg, Germany, 2001. (12) Liang, M. N.; Smith, S. P.; Metallo, S. J.; Choi, I. S.; Prentiss, M.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13092−13096. (13) Dorobantu, L. S.; Bhattacharjee, S.; Foght, J. M.; Gray, M. R. Langmuir 2009, 25, 6968−6976. (14) Camesano, T. A.; Liu, Y.; Datta, M. Adv. Water. Resour. 2007, 30, 1470−1491. (15) Hjertén, S.; Elenbring, K.; Kilar, F.; Liao, J. L.; Chen, A. J. C.; Siebert, C. J.; Zhu, M. D. J. Chromatogr. 1987, 403, 47−61. (16) Lidstrom, M. A.; Konopka, M. C. Nat. Chem. Biol. 2010, 6, 705−712. (17) Oukacine, F.; Garrelly, L.; Romestand, B.; Goodall, D.; Zou, T.; Cottet, H. Anal. Chem. 2011, 83, 1571−1578. (18) Petr, J.; Jiang, C. X.; Sevcik, J.; Tesarova, E.; Armstrong, D. W. Electrophoresis 2009, 30, 3870−3876. (19) Armstrong, D. W.; Schulte, G.; Schneiderheinze, J. M.; Westenberg, D. J. Anal. Chem. 1999, 71, 5465−5469. (20) Horká, M.; Horký, J.; Matoušková, H.; Šlais, K. Anal. Chem. 2007, 79, 9539−9546. (21) Yu, L.; Li, S. F. Y. J. Chromatogr., A 2007, 1161, 308−313. (22) Armstrong, D. W.; He, L. F. Anal. Chem. 2001, 73, 4551−4557. (23) Rodriguez, M. A.; Lantz, A. W.; Armstrong, D. W. Anal. Chem. 2006, 78, 4759−4767. (24) Tong, M. Y.; Jiang, C. X.; Armstrong, D. W. J. Pharm. Biomed. Anal. 2010, 53, 75−80. (25) Oukacine, F.; Quirino, J. P.; Garrelly, L.; Romestand, B.; Zou, T.; Cottet, H. Anal. Chem. 2011, 83, 4949−4954. (26) Berthod, A.; Rodriguez, M.; Armstrong, D. W. Electrophoresis 2002, 23, 847−857. (27) Torimura, M.; Ito, S.; Kano, K.; Ikeda, T.; Esaka, Y.; Ueda, T. J. Chromatogr., B 1999, 721, 31−37. (28) Hoerr, V.; Ziebuhr, W.; Kozitskaya, S.; Katzowitsch, E.; Holzgrabe, U. Anal. Chem. 2007, 79, 7510−7518. (29) Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. Short Protocols in Moleular Biology; Wiley: New York, 2002. (30) Destoumieux, D.; Bulet, P.; Strub, J. M.; van Dorsselaer, A.; Bachère, E. Eur. J. Biochem. 1999, 266, 335−346. (31) Gachkar, L.; Yadegari, D.; Rezael, M. B.; Taghizadeh, M.; Astaneh, S. A.; Rasooli, I. Food Chem. 2007, 15, 898−904. (32) Shen, Y. F.; Smith, R. D. J. Microcolumn Sep. 2000, 12, 135−141. (33) Shen, Y. F.; Berger, S. J.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2154−2159.

chloride ion in LE (in absolute value), ELE,itp is the electric field in the leading compartment during ITP. During tbacteria,itp, the cationic band is displaced by a distance xcat,itp given by: xcat,itp =

μcat+,LEd |μCl−,LE|

(2)

where μcat+,LE is the effective mobility of the cationic molecule in LE. Note that xcat,itp is independent of ELE,itp and therefore of the current set for the ITP process. During the CZE step, the cationic band should be displaced by a distance xcat,cze, given by: xcat,cze = L − d − xcat,itp −

L inj 2

(3)

where L is the total capillary length and Linj is the injected cationic plug length. The time required for the CZE step is thus given by: L inj

L − d − xcat,itp − 2 tcat,cze = μcat+,LEELE,cze

(4)

where ELE,cze is the electric field in LE during the CZE step. Inserting eq 2 in eq 4

tcat,cze =

⎡ ⎛ μcat+,LE ⎞⎤ ⎟⎥ − L inj 2⎢L − d⎜⎜1 + |μCl−,LE| ⎟⎠⎥⎦ ⎢⎣ ⎝ 2μcat+,LEELE,cze

(5)

For example, in the experiments shown in Figures 3 and 4, the injected sample length of benzylamine and G2 is approximately 0.143 m, the total capillary length L is 0.95 m, the distance d from the cathodic end of the capillary to the meeting point is 0.42 m, the electric field EEL,cze is 31580 V m−1, the absolute value of effective mobility of chloride ions in the leading electrolyte |μCl−,LE| is 76.17 × 10−9 m2 V−1 s−1, and the effective mobilities of benzylamine and G2 are respectively 31.86 × 10−9 m2 V−1 s−1 and 49.71 × 10−9 m2 V−1 s−1. Thus, by using eq 5, tcat,cze for benzylamine and G2 are respectively 4.68 and 1.96 min.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +33-4-6714-3427. Fax: +33-4-6763-1046. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.C. gratefully acknowledges the support from the Institut Universitaire de France and from the Région LanguedocRoussillon for the fellowship “Chercheurs d’Avenir”. We also thank COLCOM for funding a Ph.D. fellowship for F.O., and the ANR Dendrimat (grant reference ANR-096MAPR-002203) and Yorkshire Forward for support through a Development Grant to Paraytec Ltd (grant reference YHF/02803/RD09).

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REFERENCES

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Study of Antibacterial Activity by Capillary Electrophoresis Using

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