APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2010, p. 3301–3307 Vol. 76, No. 100099-2240/10/$12.00 doi:10.1128/AEM.00532-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Bacterial Capture by Peptide-Mimetic Oligoacyllysine Surfaces�

Shahar Rotem, Nili Raz, Yechezkel Kashi, and Amram Mor*Department of Biotechnology and Food Engineering, Technion—Israel Institute of Technology, Haifa, Israel

Received 28 February 2010/Accepted 23 March 2010

Most procedures for detecting pathogens in liquid media require an initial concentration step. However,poor recovery efficiencies of conventional methods, such as filtration, often lead to low sensitivity. Here, wedescribe a strategy for concentrating bacteria using their binding affinity for an oligoacyllysine (OAK), a novelpeptide-mimetic antimicrobial compound. We show that the resin-linked OAK (ROAK) efficiently captures avariety of pathogens in different media, upon brief incubation with ROAK beads or after continuous flowthrough a ROAK-packed column. Using Escherichia coli expressing green fluorescent protein, we show thatbinding occurs rapidly during incubation and persists after filtration as visualized by confocal microscopy. Thehigh binding affinity of bacteria was confirmed by surface plasmon resonance technology using an OAK-linkedchip. ROAK-bound bacteria remained viable and were readily identifiable by real-time PCR after ethanolelution. A single ROAK bead is estimated to capture about 3,000 bacterial cells in culture medium, incontaminated saline or tap water. ROAK beads can be regenerated for multiple uses after brief ethanoltreatment. Collectively, the data support the notion that OAK-based coating of polymeric surfaces mightrepresent a useful means for medium filtration as well as for concentration of bacteria.

The first step in detection of waterborne pathogens is usuallyconcentration. Concentration is necessary because the ambientdensities of pathogens in water are usually less than the limit ofdetection, and concentrating large volumes may compensatefor spatial and temporal variations in pathogen occurrence.The membrane filter (MF) technique is usually the method ofchoice for the analysis of total coliforms or of specific patho-gens from potable water. The MF method is rapid and simpleand yields definitive results. However, factors such as elevatedturbidity (23) and membrane filter type (25, 33) may severelyinfluence the sensitivity of the procedure.

Recently, there has been an emphasis on the development ofhigh-throughput assays using, for example, real-time PCR(RT-PCR), microarrays, or immunofluorescent methods.However, these rapid procedures usually require extensivesample preparation steps (3, 4, 13, 36, 39, 40), including con-centration. Microorganisms can be concentrated and sepa-rated from their constituent matrix components in a number ofways (1, 14): Whole bacterial cells have been isolated fromfood using reagents such as hydroxyapatite (5). Antibodiescoupled to magnetic beads were used to separate specific or-ganisms from human fluids, food, and water and are widelyused in different applications (8, 9, 18, 37). Novel methods forsemispecific capture of microorganisms using cell surface-de-rived lectins and carbohydrates have been proposed (10, 24).Specific capture of Mycobacterium in milk was attempted usingpeptide conjugation to a polymer (34). Furthermore, antimi-crobial peptides linked to surfaces were used for killing (20),immobilization (19), and detection (22) of bacteria. Similarly,semiautomated methods include an entire field-based process

to lyse bacteria, purify and label nucleic acids, and detectorganism signatures using microarrays (2). Also, work has beenconducted to automate separation of DNA and whole cellsfrom soils, sediments, food, and water (11, 12).

Despite the ongoing safety measures, microbial contamina-tion of drinking water can put water consumers at risk even atlow concentrations (15, 21). By the time that water analysis iscarried out and the contamination is detected, the contami-nated water will be well on its way to consumers. In addition,the small volume of sampled water (typically 100 ml) might notguarantee the safety of drinking water (15, 29). Ironically, even100 ml water might be considered a large amount to analyze, asits concentration requires either a filtration or a centrifugationstep(s), each bearing such drawbacks as problems linked toobstruction and/or use of heavy equipment, respectively (7).Emergence of new rapid detection methods is also linked tothe development of new concentration methods. For example,immunological or PCR-based methods are used after an incu-bation step because of a minimal detection limit, which ham-pers the process of rapid detection (6). Moreover, the recoveryefficiencies of conventional filtration methods are often lowand variable because of parameters related to the water (i.e.,turbidity, sediments, etc.), the bacteria (physical condition),and the filter (inhibition of microbial growth at grid lines,abnormal spreading of colonies, nonwetted areas, brittle-ness, severe wrinkling, and decreased recovery), all of whichcan lead to false-negative and poorly reproducible results (6, 7,30). There is thus a need for rapid and robust methods forconcentrating bacteria from large volumes of water with highand reproducible efficiency to detect and quantify pathogens.(21, 29, 30, 35).

Oligoacyllysines (OAKs) are a novel group of antimicrobialcopolymers (27), composed of tandem repeats of acyllysines,designed to mimic the primary structure and function of nat-ural antimicrobial peptides (AMPs). While not fully under-stood, the mode of bactericidal action of most AMPs is be-lieved to proceed via a two-step mechanism involving an initial

* Corresponding author. Mailing address: Laboratory of Antimicro-bial Peptides Investigation (LAPI), Department of Biotechnology andFood Engineering, Technion—Israel Institute of Technology, Haifa,Israel. Phone: (972) 4-829-3340. Fax: (972) 4-829-3399. E-mail: amor@tx.technion.ac.il.

� Published ahead of print on 2 April 2010.

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high-affinity interaction with a bacterial external membrane(s)followed by an internalization process that eventually leads tocell death. By linking the antimicrobial OAK to an inert resin,we hoped to be able to exploit one attribute (the bindingaffinity) and eliminate the other (the killing effect). To test thishypothesis, we produced various OAK derivatives covalentlylinked to a polystyrene-based resin via the C terminus. Pre-sumably, this construct should restrain the ability of the resin-linked OAK (ROAK) to interact with internal targets butshould not alter its properties of binding to superficial compo-nents of microbial cells.

The objective of this study is thus to evaluate the ability ofthe ROAK concept to address some of the above-mentionedflaws, using a two-step approach including (i) an initial struc-ture-activity relationship study for selection of a prototypicalconstruct for the evaluation of resin binding as an effectivemethod for neutralizing bactericidal effects of OAKs whileretaining capture capacity and (ii) evaluation of the potentialapplication of ROAKs for quantitative capture and detectionof bacteria.

MATERIALS AND METHODS

ROAK preparation. Peptides and OAKs were synthesized by the solid-phasemethod (16) using 4-methylbenzhydrylamine resin with a diameter range of 50 to100 �m (Novabiochem), applying the N-(9-fluorenyl)methoxycarbonyl (Fmoc)active ester chemistry as described previously (27). After synthesis, the resinswere deprotected by incubation in dichloromethane-trifluoroacetic acid (50:50)for 15 min at room temperature, washed twice with dichloromethane and twicewith ethanol, placed under vacuum for 3 h, and stored at �20°C. Prior to use theresin beads were washed with saline. Peptide and OAK sequences were verifiedpostsynthesis after cleavage of an aliquot, followed by liquid chromatography-mass spectrometry (LC-MS) analysis. For antimicrobial assays the crude com-pounds were purified to chromatographic homogeneity in the range of �95% byreverse-phase high-performance liquid chromatography (HPLC) (LC-MS Alli-ance-ZQ Waters). HPLC runs were performed on a C18 column (Vydac) with alinear gradient of acetonitrile in water (1%/min); both solvents contained 0.1%trifluoroacetic acid. The purified compounds were subjected to mass spectrom-etry analysis in order to confirm their composition and stocked as lyophilizedpowder at �20°C. Prior to testing, fresh solutions were prepared in water, brieflyvortexed, sonicated, centrifuged, and then diluted in the appropriate medium.

Bacterial strains. Staphylococcus aureus ATCC 25923, Enterococcus faecalis(ATCC 29212), Escherichia coli ATCC 35218, and Vibrio cholerae serotype O1,biotype Inaba (ctxA�), were grown aerobically in Luria-Bertani broth (LB broth;Sigma Chemical Company, St. Louis, MO) at 37°C with shaking overnight (16 h).

Antibacterial assays. Initial antibacterial screening was performed with fourbacteria, Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 9027),Bacillus cereus (ATCC 11778), and Staphylococcus aureus (ATCC 25923), grownin LB medium. MICs were determined by microdilution susceptibility testing in96-well plates as detailed elsewhere (28). Statistical data for each experimentwere obtained from at least two independent assays performed in duplicate.

Capture assay. For routine assays, various concentrations of bacteria in 500 �lLB were incubated in two-compartment test tubes separated by a 10-�m-cutoffmembrane (Whatman polypropylene mesh VectaSpin Micro) with OAK-coatedand uncoated beads (1.8 mg each). After 30 min of incubation at room temper-ature under shaking, the samples were centrifuged at 21,000 � g (5 min) toseparate beads from unbound bacteria and further analyzed as follows. To assessbacterial binding to the beads, the original media and filtrates were subjected todirect counts using the Live/Dead BacLight kit (Invitrogen) and to serial 10-folddilutions for plating on LB agar plates. Cell counts were determined afterovernight incubation at 37°C using the drop plate method (three 20-�l dropsonto LB agar plates). For kinetic studies, resins were incubated with 1 � 106

CFU of E. coli in LB for up to 15 min, filtered after the indicated time periods,and then plated for CFU counting.

The depletion experiment was performed essentially as described above usingsuccessive incubation-filtration cycles. During each cycle, the resin was incubatedwith 1 � 106 CFU of E. coli and filtered via centrifugation before being exposedagain to another 1 � 106 CFU.

ROAK column. For column filtration, the ROAK beads (10 mg) were packedin a glass pipette (topped by fiberglass to secure the resin). Various volumes ofcontaminated tap water (containing 102 to 105 CFU of V. cholerae) were passedthrough the column at a flow rate of 50 ml/min. Bacteria were eluted with asolution of 70% ethanol in water (1.8 ml) passed through the column, collectedinto an Eppendorf microtube, and analyzed by real-time PCR (RT-PCR).

Real-time PCR detection of V. cholerae. For DNA preparation, bacterial cellswere eluted from the resin with 1.8 ml of a 70% ethanol suspension followed byaddition of 60 �l of 3 M sodium acetate, incubated for 1 min in liquid nitrogen(or for 20 min at �80°C), and centrifuged (18,000 � g) for 15 min. The pellet wassuspended in 20 �l dilute TE buffer (1 mM Tris in 0.1 EDTA, pH 8.0), and DNAwas submitted to real-time PCR analysis as detailed below. Universal 16S rRNAprimers were selected from a conserved bacterial region to give a PCR productof 180 bp (suitable-length product for real-time PCR). Primer sequences were asfollows: UNI-F, 5�-AGGATTAGATACCCTGGTAGT-3�, and UNI-R, 5�-CGAATTAAACCACATGCTCCA-3�.

OmpW PCR primers were designed on the basis of the ompW sequence,uniquely present in V. cholerae, to generate amplicons of 588 bp from all V.cholerae strains (26). The species-specific primers were OmpW 1-5 F, 5�-CACCAAGAAGGTGACTTTATTGTG-3�, and OmpW 1-5 R, 5�-GGAAAGTCGAATTAGCTTCACC-3�.

Primers for the cholera toxin gene type A (ctxA) were selected to give a PCRproduct of 301 bp (32). Primer sequences are as follows: ctxA-F, 5�-CTCAGACGGGATTTGTTAGGCACG-3�, and ctxA-R, 5�-TCTATCTCTGTAGCCCCTATTACG-3�.

Confocal fluorescent microscopy. To visualize bacterial binding, resins wereincubated with 1 � 106 CFU of green fluorescent protein (GFP)-labeled E. coliKL2 (pSMC2 in E. coli DH5� was kindly provided by R. Kolter, HarvardMedical School, Boston, MA) (38) for 30 min and then filtered by centrifugationas described above. Resin and filtrates were transferred to microscope slides andexamined under a confocal fluorescent microscope (Nikon) using laser emissionsuitable for GFP (excitation, 485 nm; emission, 535 nm) before and after filtra-tion.

Real-time bacterial binding using surface plasmon resonance (SPR) technol-ogy. Purified resin-free OAK was immobilized on the CM5 sensor chip (BIAcore,Uppsala, Sweden) via the terminal carboxyl group using 2-(2-pyridinyldithio)ethaneamine (PDEA). Carboxyl activation was achieved as follows: 0.1 ml of 0.1M morpholinoethanesulfonic acid (MES) buffer at pH 5.0 was used to solubilize0.1 mg OAK. The solution was mixed with PDEA to a final concentration of 22mM and with ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to a finalconcentration of 13 mM, incubated for 1 h on ice, and then placed in a dialysistube (floating in buffer) to remove the excess reagents. In parallel, a 1:1 solution(20 �l) of EDC [0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in wa-ter] and NHS (0.1 M N-hydroxysuccinimide in water) was injected at a flow rateof 10 �l/min, to activate the chip surface. Next, a 30-�l solution of cystamine wasinjected to introduce a disulfide group. The disulfides were reduced with 30 �lDTE (0.1 M dithioerythritol or dithiothreitol in 0.1 M sodium borate, pH 8.5).Next, the dialyzed OAK solution (60 �l containing 10 �g) was injected toimmobilize the OAK while excess reactive groups on the chip were deactivatedwith a 40-�l solution of PDEA [20 mM 2-(2-pyridinyldithio)ethaneamine and 1M NaCl in 0.1 M sodium acetate, pH 4.0].

Bacterial binding to the OAK was determined by surface plasmon resonance(SPR) using the optical biosensor system BIAcore 2000 (BIAcore, Uppsala,Sweden). The experimental procedure and data interpretation were performedessentially as described elsewhere (17). Briefly, to monitor bacterial binding, 100�l of E. coli in saline (at 103, 104, 105, and 106 CFU/ml) was injected over theOAK-coated chip at a flow rate of 20 �l/min and collected (by an integratedsample collector) after each run. Aliquots from each run were plated on LB agarplates for enumeration after overnight incubation at 37°C. Bound bacteria wereestimated by comparing the CFU counts before and after each run.

RESULTS

Structure-activity relationship study. Because the octamerOAK derivative C12K-7�8 displayed potent bactericidal prop-erties in a previous study (27), ROAK beads carrying thiscompound as well as a series of analogs were initially screenedfor bacterial binding abilities using the capture assay (depictedin Fig. 1). As shown in Table 1, when exposed to E. coli in LBmedium, ROAK beads bearing the C12K-7�8 sequence wereable to capture �103 CFU/mg ROAK. Deleting all the acyl

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residues, which resulted in a polylysine octamer, had no sig-nificant effect, including when the peptide sequence was elon-gated to include 15 residues, supporting the notion thatbacterial capture is predominantly based on electrostatic inter-actions. Interestingly, however, deleting only the N-terminallauryl reduced the number of bacteria captured (as well asreducing the antibacterial properties of the resin-free OAK),reflecting the importance of hydrophobicity. Indeed, replacingthe aminocaprylic backbone with aminolauryl moieties gener-ated an OAK (K-7�12) that was virtually devoid of antibacte-rial activity (MIC, �50 �M) but that displayed a 1,000-fold-higher bacterial binding capability (compared to C12K-7�8).This ROAK was subjected to further characterization.

Characterization study. Characterization of bacterial cap-ture by the K-7�12 ROAK was carried out under both incuba-tion and continuous-flow conditions. In the incubation assays,binding was initially assessed through direct and CFU countsof the original inoculated bacteria and the filtrates. Subse-quently, a depletion experiment was performed to verify thebinding capacity and efficiency. Ultimately, these experiments

were repeated in the presence of different bacteria to deter-mine specificity. Figure 2a depicts a representative experimentfor the compounds listed in Table 1. Maximum load was ob-served with filtrates resulting from incubation with up to 106

bacteria/mg ROAK, but filtrates from a higher inoculum (107

and 108 bacteria/mg ROAK) displayed progressively lowerbinding proportions, reflecting gradual saturation. Bacterialbinding was virtually abolished when OAK-free beads wereused (data not shown) or with the use of ROAK beads wherethe lysine side chains were protected by a Boc [(tert-butyloxy)-carbamate] group. The fact that bacterial binding occurredonly with the deprotected form of the ROAK demonstratedthat the process was specifically related to the OAK properties.

Bacterial binding was spontaneous and rapid using eitherpolylysine (K15) or OAK-linked beads (Fig. 2b), although neg-ative cultures (binding of 106 bacteria) were obtained only withROAK beads after 60 s of incubation. No bacterial adhesionwas recorded on the control resin beads even after 30 min ofincubation.

The binding capacity of the ROAK was confirmed with adepletion experiment using consecutive incubation/filtrationcycles (Fig. 2c), where negative cultures were obtained withfiltrates resulting from the first two cycles whereas the thirdand fourth cycles yielded 7 � 105 and 1 � 106 CFU/ml, re-spectively, pointing to a gradual saturation of the ROAK bind-ing sites. From the combined data (Fig. 2a and c), we estimatethat 1 mg of ROAK (i.e., �300 beads with an average OAKdensity of 1.2 � 1012 molecules/�m2) binds slightly more than1 � 106 CFU of E. coli.

The binding efficiency of the ROAK was determined withthe use of the Live/Dead BacLight kit. Gradually increasingconcentrations of E. coli were loaded on 1.8 mg of ROAK, andthe binding yield was calculated by direct counts of the filtrateand the original inoculum (Fig. 2e). Note that filtrates with100% binding did not contain any bacteria, but the other fil-trates displayed up to 4% dead bacteria, similarly to the orig-inal suspension. Bacterial binding to ROAK was not specific toa given bacterial type, as adhesion levels were basically similarbetween typical Gram-positive (E. faecalis and S. aureus) andGram-negative (E. coli and V. cholerae) bacteria (Fig. 2d).ROAK binding was measured using both defined (E. faecalis,S. aureus, E. coli, and V. cholerae) and undefined (sewage waterafter treatment) bacterial communities (Fig. 2f). In both sys-tems the maximal bacterial load was 106 bacteria/mg ROAK.Bacterial binding could be visualized when using green fluo-

FIG. 1. ROAK design and experimental apparatus. Shown is themolecular structure of K-7�12 linked to a polystyrene bead. Under-neath is a cartoon illustration of the capture experiment: bacteria(small circles) incubated with ROAK beads (large circles) were drivento translocate across the membrane by centrifugation.

TABLE 1. Biophysical properties of OAKs and polylysines and their bacterial capture capacities

Sequence Designation No. ofresidues Qa Hb

MIC (�M) for speciesc: Bound E. coli(CFU/mg resin)d

E. coli P. aeruginosa S. aureus B. cereus

LK-cKcKcKcKcKcKcKa C12K-7�8 16 8 47.5 3.1 6.2 50 12.5 (1 0.5) � 103

KKKKKKKKa Poly-K8 8 9 20 �50 �50 �50 �50 (9 1) � 102

KKKKKKKKKKKKKKKa Poly-K15 15 16 22 �50 �50 �50 �50 (1 0.6) � 103

K-cKcKcKcKcKcKcKa K-7�8 15 9 34 �50 �50 �50 �50 (2 0.6) � 102

K-lKlKlKlKlKlKlKa K-7�12 15 9 50 �50 �50 �50 �50 (1 0.3) � 106

a Molecular charge at physiological pH.b Estimated hydrophobicity (percent acetonitrile eluent) as determined by reverse-phase HPLC.c Minimal concentration that induced 100% inhibition of proliferation after 24 h of incubation. Note that MICs of C12K-7�8 are from reference 27. Values represent

the means from two independent experiments performed in duplicate (the absence of the standard deviation reflects consistency).d Bacterial binding assessed after 30 min of incubation as described in Materials and Methods. L, lauryl; K, lysyl; c, aminocaprilyl; l, aminolauryl; a, amide.

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rescent protein (GFP)-expressing E. coli as analyzed by fluo-rescence confocal microscopy. As shown in Fig. 3b, bacteria inthe surrounding medium did not adhere to the control (pro-tected) beads but clearly interacted with the ROAK beads(Fig. 3c), indicating that bacterial adhesion occurs prior tofiltration. Bacteria remained attached to the ROAK after thefiltration step as shown in Fig. 3d, consistent with high bindingaffinity. Ethanol treatment resulted in dissociation of theROAK-bound bacteria (Fig. 3e), suggesting that ROAK beadscan be readily recycled (as confirmed below).

Most ROAK-bound bacteria were alive, as shown in Fig. 3e,f, and g, and have excluded the dye propidium iodide. Thefraction of dead bacteria (colored red) did not exceed that ofa control suspension (up to 4% as assessed by the BacLight

FIG. 3. Visualization of ROAK-bound bacteria. (a to d) Represen-tative fluorescence confocal microscopy images of GFP-expressing E.coli using the capture assay. (a) Protected (control) ROAK beadsbefore filtration. (b to d) Deprotected ROAK bead before filtration(b), after filtration (c), and after 5 min of treatment with 70% ethanol(d). Note that the residual green color on the beads is likely to begenerated from diffuse GFP emanating from ethanol-induced lysis ofbacteria. (e to h) Representative images of ROAK-bound bacteriaafter propidium iodide treatment. (e) Low-magnification image. Greenand red colors indicate live and dead bacteria, respectively. (f and g)Two successive zoom views that highlight a representative ROAK beadfrom panel e before treatment with bactericidal dermaseptin. (h) Thesame bead after treatment with bactericidal dermaseptin. (i to l) Bac-terial capture and viability. Shown are CFU resulting from the captureassay followed by plating of beads (j and l) and filtrates (i and k) on LBagar using deprotected (i and j) and protected (k and l) beads.

FIG. 2. Characteristics of bacterial binding to K-7�12 ROAKs. (a)CFU count obtained from capture assay filtrates, after incubation withprotected (gray bars) and deprotected (white bars) ROAKs, using thespecified E. coli inocula. Error bars (a, c, d, and f) represent standarddeviations from the means obtained from at least 4 independent ex-periments performed in duplicate. An absence of bars indicates con-sistency. Zero inoculum values indicate negative cultures (i.e., 10CFU/ml). (b) Time dependence of E. coli capture. CFU count wasdetermined in filtrates after incubation for the specified time periodswith deprotected (squares) and protected (circles) ROAKs. K15 beads(triangles) were used as a control. (Inset) Low-concentration experi-ment comparing K15 (triangles) and the control resin (circles). (c)Depletion experiment to determine the capacity for binding to ROAKbeads after repeated incubation/filtration cycles. Protected ROAKs(PR) were used for a one-cycle control experiment. (d) Assessment ofbinding specificity using protected (gray bars) and deprotected (whitebars) ROAKs assayed as in panel a. E.c, E. coli; V.c., V. cholerae; E.f.,E. faecalis; S.a., S. aureus. (e) Assessment of binding yield using directcounts of elevated numbers of bacteria before and after passagethrough ROAK beads. (f) Binding of bacterial communities. CFUcounts from capture assay filtrates after incubation with protected(gray bars) and deprotected (white bars) ROAKs, using the specifiedinoculum of defined bacterial mix (E. coli, V. cholerae, E. faecalis, andS. aureus, at equal concentrations; nonstriped bars) or undefined mi-crobial mix (treated sewage water; striped bars).

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Live/Dead kit). This fraction significantly increased (Fig. 3h)upon exposure to the AMP dermaseptin, known for its rapidbactericidal properties (23). Further support for this notion isprovided in Fig. 3i to l, showing bacterial growth on agar platesonly on the OAK-linked resin. The opposite outcome wasobserved with the control resin.

Binding properties. Bacterial binding under continuous-flowconditions was assessed with two different experimental set-tings using either surface plasmon resonance (SPR) or real-time PCR technologies, designed to corroborate the bindingaffinity and the potential usefulness of the ROAK concept,respectively.

For real-time monitoring of bacterial capture, we developeda model system based on the SPR technology that normallyenables binding measurements between immobilized receptormolecules and soluble ligands (17, 31). Here, we immobilizedthe OAK onto the sensor chip, bacteria were injected over theOAK surface, and their binding was monitored online. Eachexperimental run included injection of a bacterial suspensionof known concentration for 10 min immediately followed by aphosphate-buffered saline (PBS) wash step. Figure 4a shows

that capped OAK did not retain bacteria while the K15-basedchip was �100-fold less efficient than the OAK-linked chip(Fig. 4b). Thus, whereas bacteria rapidly accumulated/associ-ated on the OAK-based chip surface throughout the injectionstage, no bacterial release/dissociation was detected at thewash stage with the exception of the highest concentrationtested (106 CFU/ml), where the wash step displayed somedissociation. This behavior is assumed to reflect saturation ofthe binding sites on the chip. To validate these data, eachinjected sample was collected at its exit from the chip com-partment and plated for CFU count (Fig. 4c). The fact thatbacteria were not detected up to 103 CFU/ml while only �10CFU were counted when 104 or 105 CFU/ml was injectedsupports the notion that nearly 100% binding was obtained atthese concentrations. Accordingly, the fact that about 90%binding was obtained when 106 CFU/ml was injected suggeststhat the chip maximal binding capacity was achieved at 105

CFU/ml, which is consistent with the sensorgram.To assess capture and concentrating capabilities in a contin-

uous-flow system, we elected to mimic a diagnostic situationseeking detection of pathogenic V. cholerae. One liter of sterilesaline inoculated with V. cholerae was passed through aROAK-packed column. ROAK-bound bacteria were elutedfrom the column with 1.8 ml ethanol, which was subjected toDNA analysis by real-time PCR. Each RT-PCR run of theeluted bacteria was confirmed with a control run with the samebacterial concentration, i.e., an RT-PCR run of 104 V. choleraebacteria in 1 liter after elution from the ROAK column with1.8 ml ethanol was confirmed with an RT-PCR run of 104 V.cholerae bacteria in 1.8 ml saline (data not shown). As shownin Fig. 5a to c, positive identification was obtained with aconcentration as low as 10 CFU/100 ml saline using universal16S RNA primers (Fig. 5a), whereas positive identification wasobtained at a slightly higher bacterial concentration (100 CFU/100 ml) when specific primers (ompW and ctx) were used for V.cholerae (Fig. 5b and c, respectively).

To simulate sensitive identification of pathogenic bacteria indrinking water containing a background level of natural micro-flora, 1 liter of tap water was contaminated with low concen-trations of V. cholerae. As shown in Fig. 5d to f, positiveidentification could be achieved with a concentration as low as100 CFU/100 ml and with even lower concentrations whenompW V. cholerae-specific primers were used (compare Fig. 5dand e). Larger volumes of up to 10 liters of contaminated tapwater were passed through the ROAK-packed column, and theeluent was tested with real-time PCR using primers for the ctxgene. As shown in Fig. 5f, positive identification could beachieved with a concentration as low as 10 CFU/100 ml. Notethat, untypically, the tap water used was of particularly poorquality, where five filters were needed to filter 100 ml tap waterbecause of particle clotting, which occurred after each �20 mlper filter, on average (data not shown), whereas �10 liters ofthe same tap water could be passed through a column packedwith 10 mg ROAK while capturing bacteria flowing through.

To assess detection of a target bacterium against a highbackground of nontarget bacteria, 1 liter of tap water wasinoculated with 100 CFU/100 ml of V. cholerae and a mix of 104

CFU/100 ml of defined (E. faecalis, S. aureus, E. coli, and V.cholerae) (Fig. 5g) and undefined (sewage water after treat-ment) (Fig. 5h) bacteria. The eluent was tested with real-time

FIG. 4. Assessment of bacterial binding using SPR technology. (a)Association/dissociation sensorgrams obtained for 106-CFU/ml sus-pensions of E. coli using chips coated with K15 (dashed and dottedline), K-7�12 (dashed line; full-scale sensorgram is shown below), andprotected K-7�12 (solid line). (b) Dose dependence of E. coli bindingto K-7�12-coated chip for suspensions containing 102, 103, 104, 105

(solid lines, form bottom to top), and 106 (dashed line) CFU/ml,displaying increasing response signals. Note that control experiments(bacteria injected over noncoated chip or saline injected over OAK orK15 chips) yielded a negligible resonance signal. (c) CFU counts ob-tained from collecting and plating each run shown in panel b. Errorbars represent standard deviations from the means. An absence of barsindicates reproducibility.

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PCR using primers for the ctx gene. The detection efficienciesof V. cholerae (determined by comparing the tested RT-PCRrun to a control RT-PCR run containing the same concentra-tion of V. cholerae) against the bacterial background were 95%and 73%, respectively.

To verify their stability, ROAK beads were subjected to 10cycles of bacterial binding followed by their release with ethanol.To assess potential degradation, the OAK was cleaved from theresin and analyzed by LC-MS. In the control experiment, thisprocedure was repeated using resin-linked polylysine beads. Fig-ure 6 depicts the resulting chromatograms. Unlike polylysine,which deteriorated over time (compare Fig. 6e and f), the OAKremained unchanged (Fig. 6a and b), while molecular mass anal-ysis (Fig. 6c and d) pointed to a lack of chemical modifications,thereby confirming the chemical stability of the OAK and thesuperior robustness of the ROAK system.

DISCUSSION

The present study showed that peptide-mimetic OAKs whichwere recently developed as bactericidal compounds (27) can—upon conjugation to a polymeric surface—efficiently capture var-ious bacteria. The specific entity targeted by the OAK is unknownat this time, but the capture is likely to be mediated by nonspecific(physicochemical) interactions with an external component(s) ofthe cell wall such as lipopolysaccharides and lipoteichoic acids inGram-negative and Gram-positive bacteria, respectively. Actu-ally, the structure-activity relationships that emerged from theinitial screen suggested that both charge and hydrophobicity areinvolved in efficient capture of bacteria by ROAK beads. Theseresults suggest that further optimization studies might reveal newOAK sequences with improved binding properties and that areeventually endowed with selectivity.

This study also showed that the ROAK system is efficient incapturing bacterial species under continuous-flow or stationaryconditions. Thus, with respect to the need for new methods forefficient detection of pathogens in large volumes of water,ROAKs seem to present several advantages. The ROAK sys-tem differs from passive concentration methods, which do notseparate bacteria from contaminants of similar sizes. This may

FIG. 5. Real-time PCR amplification for the detection of V. chol-erae O1. (a to c) V. cholerae O1 (102 to 104 cells) in 1 liter saline wasrun through a ROAK-packed column and eluted with 1.8 ml 70%ethanol. After DNA extraction, 25% of cells were amplified. Shownare relative SYBR green fluorescence developments as a function ofcycle number. Samples were amplified using 16S universal primers(UNI) for detection of bacteria (a) and V. cholerae-specific primers,ompW (b) and ctx (c) locus primers. (d to f) V. cholerae O1 in tap waterwas analyzed as described above. (d and e) Analysis of 1 liter tap waterwhen amplified with ompW and with ctx, respectively. (f) Results ob-tained with 10 liters tap water inoculated with 103 V. cholerae O1 cells(10 CFU/100 ml) and amplified with ctx locus primers. NTC is anontemplate control. (g and h) V. cholerae O1 detection against a highbackground of bacteria. Analysis of 1 liter tap water inoculated with100 CFU/100 ml of V. cholerae and of a mix of 104 CFU/100 ml definedbacteria (E. faecalis, S. aureus, and E. coli) (g) and undefined bacteria(sewage water after treatment) (h), when amplified with UNI and withctx locus primers. In all panels, an absence of the second (duplicate)line indicates consistency.

FIG. 6. OAK versus peptide stability. Beads coated with K-7�12 orpolylysine-K15, exposed to consecutive cycles of bacterial binding andrelease with ethanol, were submitted to a cleavage reaction followed byLC-MS analysis. (a and b) Resulting HPLC chromatograms of thecleaved OAK before (a) and after (b) 10 cycles. Arrows indicate theOAK UV-absorbing peak. (c and d) Mass spectrometry analyses ofthe above peaks in panels a and b, respectively, for z � 3, 4, and 5. (eand f) Resulting HPLC chromatograms before (f) and after (e) 10cycles of the cleaved K15. The arrow indicates the K15 UV-absorbingpeak.

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lead to blockage and/or inhibition of the detection systemssuch as PCR. ROAK beads concentrate bacteria through cap-ture, i.e., a selective concentration system that significantlyprevents blockage. As demonstrated in Fig. 5, large volumes(�10 liters) could be rapidly filtered through a ROAK-packedcolumn, allowing high-sensitivity detection of low concentra-tions of a pathogen (i.e., 10 CFU per 100 ml). Also, consistentwith high binding affinity, bacterial capture by OAK-linkedsurfaces (e.g., polyethylene/polystyrene in beads and carboxy-methyl dextran in chips) was found to occur rapidly, and yetthe bound bacteria could be readily eluted. Such attributesmight be exploited to endow flexibility for multiple filtrationsand recycling (e.g., to reduce costs). The fact that ROAKbinding capacity was not altered and that OAK did not un-dergo chemical modifications when submitted to multiple cy-cles of bacterial binding supports this view.

In conclusion, the data support the notion that OAK-coatedsurfaces might be useful in filtration/concentration of micro-organisms from various liquid media for the detection and/ordepletion of bacteria.

ACKNOWLEDGMENTS

We thank Yael Danin-Poleg, Technion, Israel, for fruitful discussionand comments.

This work was supported by The Israel Science Foundation (A.M.,grant no. 283/08) and NATO (Y.K., project CBD.MD.981456).

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