1-Hexyl-2-cyanoacrylate compound (Neucrylate) bactericidal properties

‘vaioi Medical Inc, San Diego, California, USA 2Depzrtmer\l of Radiology, UCSD Medical Center, San Diego. Calilofriia, usA ciirrespoiiiieiise to Dr P nieiimai, vaioi Medical, 5749 Top Gun St, ste toe, San oiego, cA 92121, usA, pinedman@vaioimedicai.tiim Received is June Zml Revised 5 August zoii Accepted it August zoii ORIGINAL HESEARCH Bas 1-Hexy|—2-cyanoacrylate compound (Neucrylate) bactericidal properties Peter Friedman,‘ Violeta Casil|as,l Charles W Kerberz ABSTRACT Introduction The need for medical grade tissue adhesives both in surgery and to treat trauma has become well established Such a device has been developed and preliminary toxicity testing completed on a compounded cyanoacrvlate (Neucrylate), and its properties have been modified so it may be used as an intravascular embolic agent. Given the high incidence of iatrogenic infections in hospital. it would be desirable to have such an implantable device that inhibits dangerous bacteria. Materials and method Seven separate cultures of common bacteria were grown and exposed to Neucrylate. The impact on the exposed microorganisms was analyzed visually as well as by means of tluorescence and optical microscopy. Results The device produced high degrees of antibacterial effect when exposed to gram positive bacteria whereas it had modest impact on gram negative bacteria INTRODUCTION cerebral berry aneurysm remains a formidable disease and is difhcult to treat.” If the diagnosis is not made in advance of its rupture, the mortality rate is >50"/o. Recently, norrsurgical endovascular treatment of aneurysms has become the standard of care. With this technique, physicians are able to place bare or bioactive platinum COll5 within the aneurysm, protecting the aneurysm from the high velocity yet of blood that ultimately causes rupture.‘ 5 unfortunately, not all aneurysms can be treated with this technique—some have wide necks, irregular shapes or are larger than the largest coils.” A liquid that completely fills an aneurysm of other than spherical geometry may achieve better and more lasting results Aside from the vascular system, other inu'a—bcidy uses can be considered if a suitable malleable tissue adhesive were to become available For example, battlefield wounds, which are hemorrhagic and usually Contaminated, could be dressed with such a device. The device would also find use in orthor pedics and in women's health (the devascularizar tion of uterine fibroids, tor example), especially it that device did not support growth of common and feared iatrogenic bacterial contamination, Unfortunately, inhospital infection remains a significant problem for all surgical procedures, especially if the organism introduced into the patient is methicillin resistant Smp/iylmccus aiireus (MRSA).° ‘° Friedman P, Casillas V, Kerber CW ./ Neurolritelvenr Surg (Z0l1l doi IO H36/neurintsurg—Z0l l—U10D94 Neucrylate (Valor Medical lnc, san Diego, Calr fornia, USA), a new generation medical liquid embolic agent based on 1-hexyl»2-cyanoacrylate plus proprietary modifiers, was designed to be used in the treatment of vascular diseases of the brain—for example, cerebral aneurysms and arte— tiovenous malformations. The cyanoacrylate based device polymerizes rapidly Wlthm the aneurysm, a process triggered by the intrinsic anionic nucleo- philes found in flowing blood and on endothelium As the device polymenzes, the formed bolus conforms to the aneurysm no matter how complex the shape, filling the aneurysm up to its neck, an exercise accomplished by molding the polymeriza- tion process in place with a balloon catheter As a result of this interface, unperturbed, non—turbu— lent blood flow dynamics may be recreated. This procedure shortens the time of embolimtion and consequently the time a patient spends on the surgery table, especially when compared with the existing methods of treatment At present, Neucry- late has completed clinical tnals as a treatment for cerebral berry aneurysms.“ cyanoacrylates have been reported as being antibacterial. Dental cyanoacrylates yielded the first observations of such antibacterial proper- ties ‘Z’” Refojo er 4/ reported using cyanoacrylates to manage corneal perforations in the 1960s and later generated similar conclusions.” *6 surgical applications of cyanoacrylates shortly followed 17 Those flrst implanted cyanoacrylates were composed or short side chain polymers (eg, methyl- and ethyl—cyanoacrylate)“‘ ‘9 but ultimately those hon-iologs were found to posses toxic side effects. The n—butyl cyanoacrylate was successfully intro duced clinically, as it was less toxic while still providing the needed properties of adhesion and short polymerization time 1” 2‘ Preliminary observations in our laboratories made us wonder whether this new compounded device (Neucrylate) could in fact meet our bacteri— cidal (or at least bacteriostatic) needs. We thus designed experiments to determine whether this criterion was met, and now report our results. MATERIALS AND METHODS Six separate experiments were designed and carried out 1. A gram rnhll/rtion query A suspension of each of the following common organisms leading to a count of 105407 colony rorming units/ml was made Est/mric/Ira mli AB1157 (wild-type), Bflcrlllri subirirrs 3510 (wild-type), Bacillus 5/flllzeml lof6 Bas sc nce ATCC#6633, I call ATCC#8739, Strip/ill/ocottus almlls ATCC#6538, Gzobatll/us szzawzhermophl/us, ATCC#7953, B111///115 array/reus ATCC#NA0002 and MRSA ATCC#33591. Each colony was created from standards obtained from commercial laboratories. The surface of at least two plates of solid Lactose Broth agar (LB agar) plates was inoculated with 100-200 iii of each microorganism suspension, spread evenly and allowed to absorb. Three separate solutions were then prepared: first was pure 1— heXyl—Z—cyanoacrylate; the second comprised the liquid addi— lives; and the third was the compounded liquid, l—h€Xyl—2— cyanoacrylate plus the additives (Neucrylate). Each of the plate surfaces was divided into sections where 0 ill (the control), 10 pl, 20 iii and 50 pl of liquid were added as droplets. The two liquids containing cyanoacrylate rapidly polymerized into small disk shape forms. The G Stczlratherma/7h/lils plates were incubated at 58°C whereas the rest of the plates were incubated at 53—37“C for 24 h. The plates were then visually evaluated for bacterial growth (table 1, figure 1), 2. Inhibition ofcstab/ished co/tmfcs query, E (0/1 and B sulazll/is were grown to oDm~o.5 (logarithmic phase—growth stage in bacterial life cycle, this multiplicative function plots cell numbers per unit time, and when plotted on semilog paper, yields a straight line curve) at 37°C. A portion (200 pl) of each culture was spread over at least two LB agar plates Colonies of 3 stilrrri//is and 15 tel. were then grown on those Petri plates until confluency reached 80—l00“/o. Increasing volumes (up to 20 pl) of Neucrylate were added as droplets to the Petri dishes on top of the bacterial colomes. The drops spread into disk shapes that rapidly polymerized. The plates then were incubated at 37‘C for an additional 24 h Plates of )5 call and B subii//is without added Neucrylate served as controls. As the inhibitory growth zone became apparent (see results), a subset of this experiment was carned out. We sampled the inhibition zone and transferred it to new plates to determine whether the inhibitory halo zone was bactericidal or simply bacteriostatic. These plates were incubated for an additional 24 h and were then evaluated visually. To compare Neucrylate’s impact on growth phase versus stationary phase of bacteria, the Neucrylate was added to bacteria populated plates grown on LB and its impact was evaluated after 24 h. As before, bacteria was grown to the appropriate stage and Neucrylate samples were dispensed, allowed to polymerize forming disks, incubated for 24 h at 37"C and visually evaluated. Table 1 Impact area of Neucrylate on the various bacteria in experiments 1 and 2 volume nl Meucryiate (iii) Bacteria lasted Ill 20 50 B sumillis ill 130 330 E cirli D 0 0 S «wells Z2 Z1 38 E stearmhelmaphl/us 201 I82 277 B azhnrpnaeux do 32 64 B spizizerrii 330 427 512 MRSA 133 312 516 int trot oi int inhibition halos measured in mfllz rho rinn oi Nelicrylnle area supiracred ironi ine iniiitiiiion areas iarner nun. values averaged irorn ins applopllate eiipenrrierris. see also iigures i and z MRSA. rneinicillin resls1am Siam//acaccu: aoreus 3 To further elucidate the etiology of bacterial inhibition, the impact if each of the three solutions was compared on separate Petri dishes freshly inoculated with bacteria to see the effect of the device during the bai:teria’s growth phase Volumes of 10, 20 and 50iil of Neucrylate, the 1—hexylr2— cyanoacrylate and the additives were applied 4 To evaluate the impact of polymerization on bacteria (E toll, B suriillis, B spiziztrui, 5 zlurelts, G slmror/lemm/rhl/1/5 and B orrepneus), Neucrylate was polymerized into small disks by placing droplets on an agar plate. Those polymerized disks were then transferred to agar plates inoculated with freshly spread microbes. 5 We next evaluated whether there was any effect of pre— polymerized Neucrylate on E tcli and B sulm//rs in the liquid phase First, aliquots of 100 pl of Neucrylate were added to volumes of 100 pl us agar in 24 well sterile plates to initiate polymerization Following complete polymerization of the device, two wells were filled with E coli suspension and two with B subiil/rs. As controls, two wells with no Neucrylate were inoculated with the liquid suspension of each bacte- rium The wells were then incubated for 17 h and the results were assessed by absorption at OD 500 using Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences, now GE Healthcare UK Ltd, Little Chalfont, Buckingliamshire, UK). 6. The final experiment was a microscopic evaluation of Neucrylate’s effects on E (0/1 and B slmll/ls using the commercially available Live/Dead Viability Kit. A Deltavisloti microscope from Applied Precision (Applied Precision inc, Issaquah, Washington, USA) running Softworx software (Creative Softworx Inc has discontinued software sales and support) provided the optical imaging. First, cultures of 3 socii‘//is and E coli were grown in LB agar at 37°C in a minimum of three phials until the concentration reached OD (A600) ~05 to ensure viability and to avoid misleading results from dormant cultures. On reaching the desired state, 300 pl oi Neucrylate were added to 3 ml of each bacterial culture. (The Neucrylate would imrnediate polymerize in the broth.) Then 10 phials were incubated at 57°C for 15 min and another 10 phials for 45 min. Controls were left to grow without the addition of the device. After the incubation, samples were collected, washed in saline and resuspended in saline containing the Live/Dead stain (Live/ Dead Bal:Light Bacterial viability kit; Molecular Probes, Invi- trogen #L7oi2, Carlsbad, California, USA‘). The cells were incubated for 15 min at ambient temperature with the stain and then placed on agarose pads in microwell chambers (Molecular Probes) and imaged/visualized in the Deltavision system. Fluo— rescein isothiocyanate and tetramethyl rhodamine iso-thiocya- nate filters were utilized for the microscopy. Similarly. phials without the Live/Dead reagent were transferred into fresh media and incubated to test for viability. RESULTS In experiments 1 and 2, the bacterial growth inhibition and inhibition of established colonies, all gram positive bacteria showed significant inhibition, No significant impact was observed on gram negative (E to/i) growth (figures 1 and 2, table 1) ‘The Live/Dead viability kit is composed iii a gleen iluorescent nucleic acid stain and a red fluorescent nliclelc acid stain The principle or ditlereniiaiiori between live and dead cells is based on the ahllliy of these sialrls to permeate into the bacteria The expected result under ine microscope shows red siain lor dead ciiluile wheleas iiie live one appears green 2 of B flledman P, Caslllas V, Kelbel CW, J News/nrerveri! Sulg (2011) doi WD 1 l35/l7EUVll7(SUFg—7Ull—0lUU54 Figure 1 In experiment 1, seven bacteria were grown and the impact of Neucrylate on them was assessed. The plates were divided into lour quadrants. Duadrant 1 was used as a control. Eluadiant 2 received ll] pl, quadrant 3 received 20 pl and quadrant 4 received 50 ul ol Neucrylate placed as droplets onto the surface. Here we show only two of the plates: E coli (gram negative, left) and E spizizenii (pram positive, right). [Please also see table 1 lor inhibition zone sizes.) The large inhibition zones around the polymerized Neucrylate are seen with B spizizerlii but not with E coli. In all cases, as the Neucrylate was dropped onto the surface of the agar plates, it formed a circular disk, polymerizing almost immediately. In the subset experiment, where we transferred material from the inhibition zone to new plates, no growth occurred, suggesting that the impact or reaction is bactericidal. The size of the affected ring varied depending on the amount of Neucrylate added as well as on the gram positive bacteria itself, including the antibiotic resistant MRSA (figure 3). Neucrylate was found to have slightly stronger antibacterial impact on B sml//is in the growth phase compared with that of the stationary phase. No apparent difference was observed comparing the impact on the corresponding phases of E call’. In experiment 3, where the Neucrylate ingredients were tested to determine the source of the antibacterial impact, we found that in all tested bacteria, the 1—hexy1—2—cyanoacrylate had Fiuure 2 Observations lrom experiment 2, showing potential inhibition of established colonies. Plate A, a control, shows even and vigorous growth of B subtillis; similarly, plate B grows a lawn nfE coli. Plate C contains the increasing volumes ol Neucrylate that were added as droplets to a B sublillis lawn—the droplets spread into disks oi increasing diameter. Inhibition zones are evident. Plate B shows the increasingly larger disks of Neucrylate that have been dropped onto die lawn of E coli. No inhibition zone is evidem. Friedman P, Casillas V, Kerber CW. J Neurulmelvellt Surg (ZDl1l, dol:1D.1l36/neurlntsurg—20l P010094 Bas comparable impact to the one observed with Neucrylate, whereas the mixture of all other components of Neucrylate had no impact on bacterial growth (table 1) In experiment. 4, where the pre—polymerized disks were placed onto growing colonies, there was less inhibition of gram positive bacteria, and for the gram negative bactena, no significant inhibition. In the subsection of this experiment, the relationship between the time of pre-polymerization Cage’ of pre-polymerized device) and antibacterial impact was evaluated. The Neucrylate was polymerized on an agar plate arid the formed disk either immediately (freshly polymerized) or after ~20 miri (‘aged’) was transferred onto inoculated plates with respected microorganisms (table 2). Inhibitory halos were observed after 24 h. In experiment 5, which was designed to test liquid phase dynamics, there was complete bacterial death both for gram positive arid gram negative bacteria by 17 h (table 3). 3ol6 Basic science Figure 3 Di special note in experiment 1, the large inhibition zone around the disk ol Neucrylate on the lawn of rviethicillin resistant Staphylococcus aureus is well seen. This observation was made 24 h lollowirig exposure to Neucrylate. In experiment 6, we evaluated the impact of Neucrylate on the bactena by means of microscopy using the Live/Dead Viability Kit In the gram positive model (B sixbril/is), we observed >80% bacterial death after 15 min of incubation and 100% after 40 min of incubation (figure 4). For the gram negative inodel (E coil). only 50% inhiiiition was toiind (figure 5) Discussion In order to satisfy a clinical need for a new and better tissue adhesive and enclovascular en-ibolizatlon agent, 1—hexylr2— cyanoaciylate was compounded and combined with proprietary stabilizers and plasticizers Benchtop testing showed that the compound (known as Ncucrylate) had desirable clinical char- actenstics: it polymerized almost immediately on contact with Table 2 Studies with pre-polymerized Neiicrylate PUB-pulymlliled s ti 5 5 lieiioryiaie IIIIPIIS sreernrlierinaimilirs nrimpliaeiis spitizenii Fresh ZDI (H8 E8 262 Aged Zfl min 242 175 B5 183 Vnllllllss iii so iii ol Nsllcrvlals were polymerized on agar plates ariil than the iiisiis were carelullv extmcted eiitiei immediately liieslii or aged 20 mm and placed on plates iiiiietiated wiiii haclzrifi Attei 2: ii the piiiies were evaluated arid iiie area ol the iiiiiiiiiiiiii halos calculated Table 3 Studies in liquid E coli 5‘ silbfl I: ueiitryiate u u U U i=ie—poiyiiierired iilettiviaie ii ii a a Control 5 I4 5.76 2 89 l 41 Experiment comiiieieri in a 2i well sterile plats Average values obtained ieaiiiiiii aiiseiiiiion at oona ailei 17 ii or irieunetien. Neiicrylate means inn iii oi Neiicrylzie added in too iii at eiiiiiiie Pre—pn|ymerIZEd irieeiis Neucrvlale adiieii In media, allowed to polymerize followed llv addillori oi ooo MI in eiiiiiiie In control experiments, on ueiiciyiaie was added to me wells, liewig ||lSl growth or eiiiiiire tissue fluids, vascular endotheliurn and blood, and formed an open pore cellular sponge The porosity of the sponge allowed ingrowth of tihroiis tissue.” To date, the system has been used to treat cerebral aneurysms and arteriovenous malformations in so patients in approved clinical safety trials.“ sadly infections in hospital have become an increasingly important and difficult to treat adverse event that may complicate any surgical procedure. It would be desirable to have an implantable device that did not support bacterial growth—or, more importantly, one that would actually inhibit such growth, especially of significant hospital pathogens. Probably the most feared hospital infection is that caused by MRSA The critical question is whether a Petci dish study can actually translate into human use. On the one hand, few interventional surgeons have seen intection of any irriplanted device, even under the less than operating room sterile conditions usually found in the angiog— raphy suite. But today. we are seeing more and more combined procedures—open surgery after endovascular treatment, for example West cf 11/ reported serious infection after aortic Figure 4 grown to DD ol about 0.3. [Al Microscopy images oi control B sulztillis shows the live bacteria to be green. [Bl Alter 15 min of exposure to Neucrylate, dead bacteria appear red. The gold panicles are the Neiicrylate. lcl Similarly, after 40 min exposure to Neucrylate, no viable bacteria remain. In experiment 5, a live/dead microscopic evaluation used a commercially available viability kit to test inhibition in a broth ol 3 sulilillis 4 ol 5 Friedman P, Casillas V, Kerber CW J Neurolnterverit Sulg (ZUH) dbl W l l35/7lEUl|lilSUlQ—2Ull—0lUU54 Figure 5 The same experiment as in figure 4 was perlormed with E coll". (Al Microscopy images oi control E EU/I hrolh show numerous live [green] and several dead (redl bacteria. tel Atter l5 mlri exposure, there was no sighitleant change. (cl Arter 40 mm of exposure, approxlmately 30% ol baclalla ave dead. replacement and Halak 1! ill showed a cluster Neil infection after endovasclilar aortic aneurysm repair 2‘ 2‘ Also, the controversy over late adverse events tollowing hydrogel coated coils used for the treatment of cerebral aneurysm has never been resolved, and may be at least in part infectious 2547 Furthermore, one cotild question whether this study is even warranted, in light or a 20 year history of publications detailing the antibacterial properties of the cyanoactylate faintly In fact, those toxicity studies previously reported almost exclusively sturlierl the short chain methyl and ethyl hnmologs. whith are known to be tissue toxic and are no longer clinically used We believe that this study woultl specifically answer questions posed by not only clinicians but also regulatory agencies. There are various exogenous pathways that may cause bacterial death. In irlany cases the target is the bacterial wall, the cytoplasmic melnhrane of the targeted cell The n'la|OI distinc- tion between the types of bacteria is hasetl on the gram stain, which also distinguishes the two rrlaiur types of bacteria based on the overall membrane composition Sorne antibacterial results are antibiotic based where the material targets bacteria specific moieties (le, peptldoglycan of the outer cell wall, metabolic pathways, pirltein synthesis pathway via transmembranal internalization followed by interaction with rlbosome, etc) leading to bacterial death.“ ln numerous cases, antibiotic based treatment fails as with time the bacteria mutates to resist) Another pathway is based on the interaction betwe n the targeting material and the bacterial membrane, whc t the material creates pores in the membrane, consequently leading to lysis and bacterial death Although bacteria often develop tesls— tance to antibacterial actions, the membrane action rarely does so. the surface defining the result. a distinction that has been correlated to the glam stain of the bacteria Interestingly, anti— bacterial results using shorter chain cyanoacrylates were previ— ously reported btit we suspect the true molecular mechanism of the activity has not been established 1‘ "’ Evaluation of colonies on the agar plates showed inhibitory action of Neuerylate on all gram positive bacteria whereas no impart was observed on the gram negative F eoli (tigures l and 2) When Netlcrylate or liquid ryanoarrylate was dropped on the agar plate, the droplet hardened (polymerized) almost lmmedr ately, creating a llat circle. in experiments 1 and 2. a region tree of bacteria in the shape of a ring appeared around the poly— merizerl device. To address whether the antibacterial effect was the result of bactericidal or bacteriostatlc impact by Neucrylate, samples from the inhibitory halo region were collected and transferred to fresh plates for growth (experiment 1B) Upon extensive incubation of those plates at ~35"C. analysis showed no bacterial growth or colony formation This finding supports the fact that Neucrylate is bactericidal. Similar results were observed in the past by others utilizing different cyanoactylate preparations ‘‘ ‘ 7” “ The size or the attected ring varierl depending on the amount or Neticrylate added as well as on the spctitit gram positive bacteria itself, including the antibiotic resistant MRSA (figure 3) table 1 summarizes the ring area (mm?) as a function of Neiierylate volume (iii) applied, and bacteria type with some bacteria, comparison between the Neucrylate, the liquid 1— hexyl-2-cyantlacrylate, and the proprietary plastlclzer and stabilizers were tested As shown in table 1, the cyanoacrylate moiety carried the sole responsibility for the antibacterial activity (the slight increase in the inhibition zone comparing Neilerylate with pilre cyanoacrylate possibly relates to the pattern of polymerization, the latter yielding a hard and dense dislr whereas the ftlrmei produces a soft and porous result) ln the case of B mlvrlllis. the inhibition zones around the polymeric disk of Neticrylate were slightly larger when the bacteria were in the log growth phase compared with the stationary phase Testing the impact of Neuctylatc on both E 5/ll7li'/lls and E (0/1 in llqulcl. the device was added to viable cells in a 24 well plate We measured tuihiclity as an indicator of viability In the case of the gram positive bacteria, the expected terminal impact was observed: the bacteria did not recover when placed in fresh media. in the case of gram negative L tell. we observed terminal impact only at higher concentrations of Neucwlatc (l00—400 ul of hacteria) Evaluating the impact of Neticrylate on the bacteria by means of microscopy using the Live/Dead viability Kit and 15 siilm//is as the gram positive model, we observed >80% bacterial death after 15mm and 100% after 40 min of Neucrylate incubated with the bacteria (figure 4). In the similar experiment with gram negative F (L7/I. some impact of Neucrylate was observed in the form of bacterial death t~5tw/ii) The lipopolysaccharido external membrane of the gram negative 1: rali may be the variable which creates resistance.” The antibacterial impact or Neiictylate increased as the amount of added Ncucrylate increased Fllcdmarl P, Caslllas \/, Kerbcr CW ./ /\/F,-llml/lll=,-ll/F,-nr Sllrg (ZUl ll (‘lfll I0 ll35’llClAlll'll5illg—Zl7l lrUlUU9/l 5 ul 5 Ba 1: sc ence Attempts to grow bacteria on prepolymerized Neucrylate led to no colony formation. Polymerizing Neucrylate into disks on agar and then transferring the polymerized disks onto bacteria colonies grown on a plate also led to formation of inhibition halos after 24 h. In the case of gram positive bacteria, based on the microscopy pictures showing death along with lack of tecovery of bacteria from the affected area on the tissue—cu1ture plate, we conclude that the antibacterial impact is bactericidal, not bacteriostatic The results are summarized in table 2 The postulate is that Neucrylate posses cacterieidal properties causing merncrane decomposition of the gram positive bacteria. The viability test or E to/l' and B slibtil/is in the liquid phase led to no bacterial growth. The results indicate a strong termi- nal impact of Neucrylate on gram positive bacteria We postulate that the close molecular proximity between the bacteria and the Neucrylate causes disruption in the bacterial wall. In contrast with the gram positive antibacterial impact, the loll of gram negative bacteria is postulated to occur upon intimate molecular association between the microbial wall and the Neutrylate, Cram positive death effects showed as an impact ring on all plates whereas the gtam negative bacteria seemed to be inhibited only on molecular association reaction cyanoacrylates pol}/— merize when in contact with an anionic initiator. Coincidentally that is the charge of the gram positive bacteria, Whether it is coincidence or mechanism is yet to be established. Our studies support the contention that this cyanoacrylate compound is bactericidal to certain bacteria, especially those that are gram positive The inhibitory effect in gram negative bacteria depends on processes and conditions that may not be found in arteries or in human wounds, importantly, its bacte- ricidal properties against MRSA is a welcome finding Acknowledgments The authors grateiully acknowledge Ms ileiia shechter. tlepanment oi Ulganlc chemistry, weizmann institute oi Science, Rehovot, lsrael, ior help designing and evaluating some oi the experiments, Pmlessui Aoraham Minsky, oepartment oi Olgaillc chemistry, weizmann institute oi science. Renovct, lsrael. ior ioluaole discussion oi the results. Ms vael Mutsaii. Department oi organic chemistry and or vladimir kiss, oepanment oi Biological Chemistry, weizmann institute oi science, Rehcvct. lsrael. ior their consultation with the microscopy. or ken Barlllal and Mt Kllk Nelson. itius Thelaneuilcs. san Diego. calilornia, ior their help with the MRSA, and Mr Mark shannon and Ms Tlacev Minutolo, Dliadlanls scientiiic Inc, san oiego, caliicrnia, ior their help with screening some oi the microorganisms Flnidilin valor Medical inc. competing interests PF and vc are employed hy vAtoR Medical inc. cwi< is a stockholder in valor Medical lnc Pwvanancs and put review itot commissioned, exlelilallv peer reviewed REFERENCES I tinglialrni G, vinueia F, sepetka l, era/. tlettrothromlzosis oi saccular arieulysms via endovascular approach J lueurosurg last 15 i—7 2 Maurice-williarns its. Aneunysm surgery aiter the lntemational sniiarachnotd Aneurysm Trial USAT) J Neuwl Iveurcsurg Psychiatry 2004,75 a07—ii 3 uloiynenir A.l, i