APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1992, p. 3157-31620099-2240/92/093157-06$02.00/0Copyright X) 1992, American Society for Microbiology

Metal-Based Formulations with High Microbicidal ActivityJOSE-LUIS SAGRIPANTI

Molecular Biology Branch (HFZ-113), Center for Devices and Radiological Health,Food and Drug Administration, Rockville, Maryland 20857

Received 1 April 1992/Accepted 23 June 1992

Substances were evaluated for their relative potencies in inactivating Junin virus, Escherichia coli, andspores of Bacillus subtilis. Under the conditions of our test, glutaraldehyde was the most efficient agent amongall substances currently recommended for disinfecting and sterilizing medical devices. Either copper or ironions by themselves were able to inactivate virus with an efficiency similar to that of substances currently usedfor disinfection and sterilization. The microbicidal effect of metals, however, was enhanced further by theaddition of peroxide. The mixtures of copper and peroxide described here were more efficient thanglutaraldehyde in inactivating viruses and bacteria. The addition of a metal chelator to metal-peroxidemixtures further increased the microbicidal potency of the reagent. The formulations described in this studyshould be harmless to people but able to quickly and efficiently inactivate microorganisms, particularly viruses.

The accumulated data indicating the presence of patho-genic organisms in medical devices after use suggest thatcurrent disinfection practices are unsatisfactory (7, 10, 30).Graft prostheses, such as dura mater, skin, cornea, and heartvalves of human origin, have been implicated in the trans-mission of disease, including Creutzfeldt-Jakob disease (aform of progressive dementia) (2, 21, 23) and AIDS (1).Transmission of infectious microorganisms by spirometersand some models of ventilators and respirators has beensuspected (17).

Persistent contamination of bronchoscopes with Serratia,Pseudomonas, Mycobacterium, and Bacillus species, lead-ing to some fatalities among patients, has been described (9,12, 18, 20, 24, 28, 29). After bronchoscopies, incidences ofpneumonia (6%), bacteremia (15%), and fever (16 to 46%)have been reported (5, 25). A sampling of a series ofbronchoscopes used on patients with AIDS showed contam-ination with respiratory-tract commensals, Candida albi-cans, hepatitis B virus, and, in all cases, human immunode-ficiency virus (HIV) (14).The use of glutaraldehyde, hydrogen peroxide, chlorine,

or formaldehyde has been suggested for disinfection (13).Mainly on the basis of experiments with chimpanzees in-fected with hepatitis B virus, the Working Party of theBritish Society of Gastroenterology recommended that con-

taminated bronchoscopes should be disinfected for 4 min in2% alkaline glutaraldehyde after cleaning (30). Since most ofthe available disinfection data are concerned with broncho-scopes, the disinfection practices for other medical devicestend to duplicate those used by the bronchoscopy units.Although glutaraldehyde is the disinfecting agent of

choice, there are still serious problems associated with itsuse. A survey of 43 endoscopy units in the United Kingdomfound that 37% of them had problems with sensitization ofstaff personnel (3). Most individuals affected are those withthe greatest exposure, that is, the endoscopists with the mostexperience. The efficacy of glutaraldehyde has also beenquestioned, since a 1% solution resulted in an ineffectiveinactivation of HIV in the presence of serum (15).

Mutagenicity and carcinogenicity have been reported forformaldehyde, and an association between the increased riskof spontaneous abortion and exposure to formaldehyde hasbeen indicated (16, 27). However, a formaldehyde-based

disinfectant is used as an alternative agent by personnel whoare sensitive to glutaraldehyde.

In spite of the drawbacks with regard to efficacy and staffsensitization, exposure to 2% glutaraldehyde for 4 to 20 minis now the disinfection method of choice for a wide variety ofmedical devices, and it is used in medical units throughoutthe world (7, 14, 30).

Several metal salts, including copper naphthenate, copper8-quinoleinate, copper octoate, and ammoniacal copper ar-senate, have been used as preservatives against fungal attackin crops, textiles, paints, and wood (for a review, seereference 32). However, neither the relative microbicidalpotency of copper ions nor their efficacy in disinfectingmedical devices has been established.

In this work, different liquid disinfecting agents werecompared for their ability to inactivate virus and bacteria.The objective of this study was twofold: (i) to establish themost-active liquid disinfecting agent among those substancescurrently in use, and (ii) to investigate whether metal-basedreagents with a microbicidal activity comparable to or betterthan that of the best disinfectant tested could be formulated.Data are presented which indicate that glutaraldehyde is themost potent disinfectant currently used and that metal-basedreagents can be formulated with a higher microbicidal activ-ity than that of glutaraldehyde.The results presented here may contribute to more effec-

tive disinfection and sterilization of medical devices. Bettersterilizing agents, harmless to people and equipment andcapable of quickly and efficiently inactivating microorgan-isms, in particular viruses, could be a major contribution tothe quality of health care.

MATERIALS AND METHODS

The model described here for assessing virucidal activitywas developed by using the attenuated strain XJ-clone 3 ofJunin virus (JV), obtained from C. J. Peters (USAMRIID,Ft. Detrick, Md.). JV is a member of the Arenaviridaefamily. Like the etiologic agents of AIDS (i.e., HIV, for-merly named lymphadenopathy-associated virus or humanT-cell lymphotropic virus), JV is an enveloped virus whoseRNA genome is surrounded by protein (4). The attenuatedstrain of JV was selected for this work because it is harmlessto humans (26).

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Vero cells, the host of JV, were obtained from theAmerican Type Culture Collection (Rockville, Md.) andgrown by the method of Bushar and Sagripanti (6). JV (5 Ru),at a concentration of 105 to 106 PFU/ml in Eagle's minimalessential medium (EMEM) containing 5% fetal calf serum(FCS), was diluted 1:4 in phosphate-buffered saline (PBS)(pH 7.4), before 8 pul of the inactivating agent at variousconcentrations was added. The total volume was adjusted to28 ,u so the assay could be used to test small volumes ofliquid remaining within the intricacies of medical devices.After a 30-min incubation period at 21°C in sterile conical1.5-ml Eppendorf tubes, the reaction mixture was diluted to1 ml with ice-cold EMEM-5% FCS and put on ice. Virus wastitrated immediately by overlaying 0.2 ml over 80% confluentVero cells in 35-mm-diameter, six-well tissue culture platesas previously described (6). Between 200 and 250 virusplaques per well were typically obtained in untreated con-trols. The assay was performed in duplicate, allowing virussurvival as low as 0.2% in untreated controls to be mea-sured.

Virus survival was studied through more than 1,000-foldby scaling up the method described above. In these experi-ments, 1.2 x 104 PFU of virus was treated with testsubstances and inoculated into 175-cm2 tissue culture flasks.The dose of sterilizing or disinfecting substance needed toinactivate 50% of virus (ID50) was used for comparison,since this parameter has been considered ideally suited toassess inactivation of HIV (22).

Kinetics experiments were done in 1.5-ml plastic tubes byadding 50 pI of virus (at about 106 PFU/ml) in EMEM-5%fetal bovine serum and 115 ,u of water, PBS (pH 7.1), orEMEM (pH 7.3)-5% FCS. Immediately after the test sub-stance was added (115 RI) at the designated concentration, a28-pA aliquot from each sample was withdrawn. This is thezero-time sample. At five additional preestablished timeintervals, 28-pI aliquots were withdrawn from each sample.To stop any reaction, all aliquots were immediately dilutedto 1 ml with ice-cold EMEM-5% FCS and kept in ice untilthe last time point was obtained. Infectious virus in 0.2 ml ofeach aliquot was titrated on Vero cell monolayers in six-wellplastic culture plates (6).For the bactericidal assay, strain NM514 of Eschenichia

coli was obtained from Amersham (Arlington Heights, Ill.)and cultivated in our laboratory. An individual colony waspicked from a stock plate of bacteria and inoculated into 10ml of Luria-Bertani broth media (LB broth) with 0.4%maltose. After incubation at 37°C overnight with shaking(200 rpm), 1 ml of culture was added to 50 ml of prewarmedLB broth-0.4% maltose. Cultures were vigorously shaken at37°C until an optical density at 600 nm of 0.75 was reached(approximately 4 x 108 cells per ml in the logarithmic phaseof growth). Bacteria (5 pA) were diluted 1:4 in PBS, and 8 pAof the test substances was added in sterile plastic Eppendorftubes. After 30 min of incubation at 21°C, samples werediluted to 1 ml with cold LB broth-0.4% maltose, diluted1:10, and plated (10-pA inoculum) onto 85-mm-diameter LBbroth-agar Lennox plates (GIBCO/BRL, Grand Island,N.Y.). Typically, about 300 colonies per dish were obtainedfor untreated controls.

Spores of Bacillus subtilis were prepared by the method ofCyr and Pollard (8) and generously supplied by H. Cyr (Foodand Drug Administration, Rockville, Md.). Approximately108 spores were exposed to the test substances in a 280-pAvolume at room temperature. At timed intervals, 28-pAaliquots were withdrawn and titrated in LB broth-agarLennox petri dishes.

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Active Substance (mg/L)FIG. 1. Comparative virucidal activities of common disinfecting

agents. Virus was incubated 30 min at 21°C in the presence of 0.9%FCS and various concentrations of the following substances: gluta-raldehyde (Gl), chlorine (as sodium hypochlorite) (Cl), hydrogenperoxide (Px), and formaldehyde (Fm). After treatment, the remain-ing infectious virus was titrated as described in Materials andMethods.

Cupric chloride (CuCl2. 2H20), nitrilotriacetic acid (dis-odium salt; NTA), and formaldehyde (37%) were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). Ferric chloride(FeCl3. 6H20) and 30% hydrogen peroxide were obtainedfrom Mallinkrodt, Inc. (Paris, Ky.). Glutaraldehyde (8%) insealed glass ampoules was purchased from Ladd ResearchIndustries (Burlington, Vt.) and diluted daily before experi-ments were performed. A commercial glutaraldehyde-baseddisinfectant (Cidex; Surgikos, Johnson and Johnson, Olling-ton, Tex.) was generously supplied by Z. Glaser (Food andDrug Administration). Chlorox was used as the source of5.25% sodium hypochlorite. Metal salts were dissolvedgenerally at 1 g/liter in 1 ml of distilled water (in some casesacidified with 10 RI of 2 N hydrochloric acid) and filteredthrough 0.22-,m-pore-size membrane units (Millex GV;Millipore, Bedford, Mass.). Inactivating agents were seriallydiluted 1:10 in sterile distilled water at neutral pH, andexperiments were performed within 3 h thereafter. Newdilutions from the stock solutions were prepared before eachexperiment. The mixtures of metal ion and peroxide areexpressed as a ratio of one to the other (each in milligramsper liter). A mixture of copper and peroxide at 1:100 corre-sponds, for example, to a proportion of 100 mg of hydrogenperoxide per 1 mg of copper (in the volume tested).

RESULTS

The results of the comparative study shown in Fig. 1indicate that glutaraldehyde is the most active liquid disin-fectant among those currently used. Indistinguishable resultswere obtained for alkaline glutaraldehyde, which was acti-vated by adding bicarbonate according to manufacturer'sinstructions (Cidex), and for glutaraldehyde, which wasbrought to pH 7.4 by buffered media immediately beforeassay.

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METAL-BASED REAGENTS WITH HIGH MICROBICIDAL ACTIVITY

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FIG. 2. Virucidal effect of copper and iron. Virus was treatedunder conditions identical to those described in the legend to Fig. 1with glutaraldehyde (Gl), copper(II)-chloride (Cu), and iron(III)-chloride (Fe), before titration. Virus survival after exposure tovarious concentrations of saline (NaCl) is included as a control.

The virucidal activities of iron [Fe(III)] and copper[Cu(II)] were compared with that of glutaraldehyde (Fig. 2).On the basis of ID50 values, copper and iron are approxi-mately 5 and 25 times, respectively, less active than gluta-raldehyde. However, the ID50s of copper and iron aresmaller than the ID50s observed for recommended disinfec-tants such as formaldehyde, peroxide, and chlorine (Fig. 1).The enhancement of the virucidal activity of copper at low

concentrations (0.05 mM = 3.17 mg/liter) by the presence ofdifferent concentrations of peroxide was assessed. The re-sults shown in Fig. 3 indicate that peroxide potentiates thevirucidal capacity of copper throughout the range of concen-trations studied. It may be valid to expect an even highervirucidal activity for mixtures with higher proportions ofperoxide to copper. In the absence of metal ions, peroxideshowed a relatively low virucidal activity (Fig. 1). Copperand peroxide in combination inactivated far more virus thanthe sum of their independent activities, i.e., they act syner-gistically.The virucidal and bactericidal activities of different con-

centrations of a mixture containing 100 mg of peroxide permg of copper were compared with those of glutaraldehyde.A virucidal ID50 for the mixture was obtained for a copperconcentration of 0.07 mg/liter, while the ID50 for glutaralde-hyde was not reached until a concentration of 2 to 7 mg/literwas used (Fig. 4A). This suggests that the mixture is roughly50 times more efficient in inactivating virus than glutaralde-hyde when the concentrations of copper and glutaraldehydeare compared.The effect of the copper-peroxide mixture for an expanded

range of virus survival is shown in Fig. 4B. Copper at aconcentration of 10 mg/liter in a ratio of 1:100 with peroxideinactivated virus by 3.5 orders of magnitude in the inoculumafter 30 min. Peroxide alone showed little inactivation.Experiments on the survival of E. coli indicated that an

ID50 was achieved for a copper concentration of 0.45 mg/literin the presence of 45 mg of peroxide per liter. In the sameexperiment, 4.6 mg of glutaraldehyde per liter was required

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0:1 " 1:1 2:1 20:1 100:1 mol:mol(1:1.9) (1.1:1) (11:1) (54:1)(mg:mg)

Ratio Px:CuFIG. 3. Enhancing effect of different peroxide concentrations in

the virucidal activity of copper. Virus was treated for 30 min at 21'Cin the presence of 3.17 mg of copper(II) (Cu) per liter and a peroxide(Px) concentration ranging from 0 to 170 mg/liter (upper horizontalaxis). The proportion of peroxide in the mixture is indicated in thelower horizontal axis. Averages (+) of duplicate experiments (@)are indicated.

to kill 50% of the bacteria after 30 min of incubation at roomtemperature (data not shown).The World Health Organization guidelines on sterilization

and disinfection methods against HIV indicate inactivationof B. subtilis as proof of sterilization (31). The concentra-tions of copper and peroxide in a mixture were adjusted toyield a sporicidal activity similar to that of 2% glutaralde-hyde (Fig. 5). Copper (0.2%) plus peroxide (5%) inactivatedspores at a similar rate as glutaraldehyde (2%). Both prepa-rations resulted in 1,000-fold inactivation after approxi-mately 35 min. A decrease greater than 5 orders of magni-tude in spore titer was observed after 60 min.Glutaraldehyde diluted to 0.2% required more than 18 h toreduce the spore titer by 1,000-fold. Copper (0.2%)-peroxideat a ratio of 1:50 decreased the spore titer 1,000-fold in 15min (data not shown). The titer of untreated spores kept inwater remained constant during the experiment (Fig. 5,control).The kinetics of virus inactivation for a mixture of copper

and peroxide (1:100) was compared with that of glutaralde-hyde. The results of three independent experiments (Fig. 6)indicate that the virus inactivation rate for a 1:100 mixture ofcopper and peroxide in the presence of 0.9% serum wasabout 4.5 times faster than that of glutaraldehyde. In mediacontaining 5% serum, the copper mixture inactivated virus

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thexais.(ATh efecs o th mitue (ix:10)4 an ltrl-01deyd (G)iOhrsnc fO9osrmarcoped(BTh _\0fterincubation with H2 alone. *\0.040.001 0.01 0.1 1 10 100 0.03 0.1 0.3 1 3 10

Concentration (mg/L) dFIG. 4. Virucidal activities of a copper-peroxide mixture.bThe

concentration of copper in a 1:100 mixture with H202 is plotted on 0Mix 100the x axis. (A) The effects of the mixture (mix 1:100) and glutaral- 10dehyde (GL) in the presence of 0.9% serum are compared. (B) The 2virucidal effect of the copper-peroxide mixture in the presence of 5%serum at the range of low virus survival is shown. A, virus survivalafter incubation with H202 alone. >

about five times faster than glutaraldehyde did (data notsshown). The high survival of infectious JV after incubation E

with saline (sodium chloride) confirms that the virucidal Eeffect of the copper salt is not due to chloride but to the metalion (Fig. 6).The stability of the reagent was tested after mixing copper

and peroxide. The inactivation rate remained constantthroughout the 22-h time span studied (data not shown).Glutaraldehyde demonstrated an identical inactivation ratewhether it was a commercially available disinfectant (Cidex)or a technical- or reagent-grade chemical. Also, an identical 1 I I

0 10 20 30 45 60

Incubation Time (minutes)0~~~~~8 A... FIG. 6. Comparative kinetics of virus inactivation. The inactiva-8 4 o .. ..-IN......................

tion ofvirus by a mixture (mix 100) of copper and hydrogen peroxide(copper chloride [1 mg/liter]; ratio, 1:100) was compared with thatby glutaraldehyde (1 mg/liter; Gl). The incubation was at room

7 temperature (21'C) in the presence of 0.9% serum, and the effect ofsodium chloride (NaCl) is included as a control.

0 6o 6-\XL \ \\inactivation rate was obtained when Cidex was alkalinizedcn Mix 1:25 2% by adding bicarbonate according to the manufacturer's in-Mix 1*25 \GL2G structions or by raising the pH to 7.4 in buffered media.

5 - The microbicidal potency of metal-peroxide mixtures wasenhanced further by a metal chelator such as NTA. Amixture of iron and peroxide (1:1) at a concentration one-fourth the ID50 was enhanced in its inactivation potency four

o to seven times by the addition of NTA (Fig. 7). The sameconcentrations of NTA and iron, either alone or mixed, didnot show virus inactivation. NTA performed best when it

3 was mixed with the metal before the infectious sample wastreated. The enhancing effect of NTA was observed in thepresence of 0.9% serum and at a neutral or slightly alkaline

2 pH.

0 10 20 30 45 60

Incubation Time (Minutes) DISCUSSIONFIG. 5. Sporicidal activity of copper-peroxide. Log of spores

surviving after various incubation periods in the presence of 0.2% Several substances have been recommended for liquidcopper-5% H202 (mix 1:25), 2% glutaraldehyde (GL 2%), or sterile sterilization, but their relative efficacies have not beenwater (A). clearly established. The assays described here allow a com-

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500

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40 Iron 1 mg/L:Peroxide 1 mg/L |

03

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0.00 0.03 0.31 3.14 31.4

NTA Concentration (g/L)FIG. 7. Enhancing effect of NTA in virucidal activity of iron ions

and peroxide. Inactivating potency is calculated as the percentage ofvirus inactivated by the mixture Fe-H202 (ratio, 1:1; 1 mg of eachper liter) in the presence of NTA divided by the percentage of virusinactivated by the same mixture in the absence of chelator. Iron waschelated to NTA at pH 7.8 prior to incubation with virus in 0.9%serum.

parison of substances with very different microbicidal activ-ities over a broad concentration range.Aqueous glutaraldehyde (2%), stabilized hydrogen perox-

ide (6 to 10%), and aqueous formaldehyde (8 to 10%) arerecommended for liquid sterilization (13). Various concen-trations of glutaraldehyde, formaldehyde (3 to 8%), andperoxide (6 to 10%) are considered to have high disinfectantactivities, while chlorine compounds (50 to 500 mg of chlo-rine per liter) are considered to have intermediate disinfect-ing activities. In this study, glutaraldehyde was the mosteffective substance among those currently used for liquiddisinfection. Other recommended disinfecting agents, suchas peroxide, hypochlorite, and formaldehyde, showed sig-nificantly less virucidal activity than glutaraldehyde did.The present work demonstrates that copper(II) and iro-

n(III) can inactivate virus. These ions inactivate virus withefficiencies 5 to 25 times lower than that of glutaraldehydebut 2 to 20 times more effectively than recommended disin-fecting substances such as formaldehyde, peroxide, or chlo-nne.

Peroxide potentiates the virucidal and bactericidal effectsof metals. This enhancement increases with the proportionof peroxide to metal in the mixture, throughout the rangetested. The enhancement by peroxide occurred in a concen-tration-dependent fashion and does not reach saturation atthe ratios studied.HIV in serum remains infectious for more than 15 min

when incubated with 1% glutaraldehyde (15). This could beimportant, since reuse of glutaraldehyde after about 20bronchoscopic procedures reduces its active concentrationto about 1% (14). Metal-peroxide formulations exhibitedmicrobicidal activities higher than that of glutaraldehydeeven in the presence of 5% serum.

More than a third of the personnel who use glutaraldehydedevelop an adverse reaction to it (3). Exposure or contact toeither copper or iron should not present such a problem.Levels of copper and iron range from 1.1 to 2.3 mg per literin normal human sera (11, 19). The amount of metal remain-ing in medical devices sterilized with either copper or ironshould be below the physiologic range; hence, the metal

should pose little or no risk to human health. This is avaluable advantage of copper- or iron-based formulationsover substances currently used in disinfection and steriliza-tion.

Furthermore, the data presented indicate that a metalchelator, such as NTA, can increase the virucidal potency ofa mixture of iron and peroxide approximately sevenfold. Thecombination of three different types of substances providesgreat flexibility in the formulation of microbicidal systems.Thus, metals alone, metals mixed with a reducing agent suchas peroxide, or metals in a ternary system formed by theaddition of a reducing agent and an adequate chelator couldbe used for the sterilization and disinfection of reusablemedical devices and graft materials. Different metals, reduc-ing agents, and chelators could be selected, and their pro-portions to each other could be varied to yield an efficientmicrobicidal formulation harmless to both personnel anddelicate medical equipment.

ACKNOWLEDGMENTS

We thank Grace Bushar, Howard Cyr, and Zory Glazer forsupplying Vero cells, B. subtilis, and Cidex, respectively, and C. D.Lytle and H. Cyr for helpful discussions of the manuscript.

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