j. Cosmet. Sci., 52, 369-375 (November/December 2001)
A modifiedcup scrubmethodfor assessingthe antibacterialsubstantivityof personalcleansingproducts WARD L. BILLHIMER,
JAN S. ENGLEHART, GAYLE Y. RAINS, and BRUCE H. KESWICK, TheProcterO GambleCompany, Cincinnati,OH 45241.
Accepted for publication August15, 2001.
An improvedin vivomethodfor evaluatingthe antibacterialsubstantivityor residualeffectiveness of bar soapsand other personalcleansingproductsis presented.The effectiveness of an antibacterialbar soap containing1.5% 3, 4, 4'-trichlorocarbanilide (TCC) versusits soapvehiclewasevaluated undersimulated conditionsconsidered optimalfor bacterialgrowth,proliferation,and possibleinfection.A washoutperiod to clearthe skin of anyantimicrobialagentspreviouslyusedwasfollowedby a treatmentperiodin which the subjectswashedoneof their forearmswith the antibacterialsoapand the other forearmwith the soap vehicle.Either immediatelyor 24 hoursfollowingthe final wash,three test siteson both forearmswere inoculated with S. aureus andoccluded with Hill Top Chamber © patches.At intervalsof 30 minutes,two hours and five hours, the patcheswere removed.The bacteria on the skin were harvestedusing the Williamson-Kligmanscrubtechnique(1,2) to determinethe numberof survivingCFUsat eachtime period. The methodsuccessfully demonstrated that sufficientTCC had remainedon the skin for 24 hoursafter the finalwashto effectivelyinhibit the growthofS. aureus on the skinfor aslongasfivehoursafterinoculation.
Skin infectionsdue to Staphylococcus aureussuch as folliculitis, furuncles,carbuncles, impetigo,and woundinfections,arerecognized asa commonhealthproblem(3,4). The regularuseof an antibacterialsoapfor personalcleansinghasthe potentialto controlthe growth of S. aureuson the skin and to aid in the preventionof thesediseases. The surfactantbaseremovesbacteriafrom the skinwhile depositingan activeingredientthat can help to control the regrowth of remaining organismsand help to prevent the colonizationof other transient,potentially pathogenicorganisms.Demonstratingthe disease preventionpotentialof an antibacterialsoapunderconsumerconditionsrequires significantlylarge numbersof subjectsand extensiveamountsof time, and it is often financiallyprohibitive.Instead,it is usefulto demonstrate the benefitsof washingwith antibacterialsoapsby using controlledclinical methodsdesignedto demonstratethe differencesbetweenplain soapand water (placebo)and the product containingan antibacterialagent. 369
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Variousclinicalmethodsfor evaluatingthe efficacyof antibacterialsoapproductshave beenreportedin the literature(1,5-7). Thesemethodstypicallymeasurethe ability of a productto reducethe residentflorathrougha continuous actionor evaluatethe ability of a productto reducethe levelof markerorganisms on artificiallycontaminated hands. Thesemethods,however,do not measurethe ability of an antibacterialagentto remain on the skin for a prolongedperiodand continuesuppressing growth of acquiredskin pathogensin situ. This antibacterialsubstantivityor residualeffectiveness is an important characteristic to measurein evaluatingthe overallperformanceof an antibacterial product. Aly and Maibach (8) reportedthe developmentof a method to evaluatethe residual effectiveness of a singleapplicationof topicalantibacterialproductsagainstvariousskin pathogens.This methodincorporatedthe occlusionof treatedskin sitesinoculatedwith bacteriaand the enumerationof the bacteriaremainingafter five hoursof occlusionvia the cup scrubtechnique.Residualantibacterialeffectiveness wasdeterminedby comparingthe differences in growthand survivalof the testorganismon sitestreatedwith eitherthe antimicrobialagentor a vehicle.Finkeyetal. (9) andScalaetal. (10) usedthis methodto evaluateantimicrobialsoapsfollowingrepeatedapplication.Thesestudiesdid not, however,establishhow quicklythe antibacterialeffectoccursor how long it lasts. Our objective was to determine the rate and duration of the antibacterialaction of
antibacterialsoaps undersimulateduseconditions. We modifiedthe previouslyreported residualeffectiveness testprocedure(9,10) by inoculatingthe siteseitherimmediatelyor 24 hoursafter the final washand harvestingthe survivingorganismsafter various intervals of occlusion.
Two studiesillustrate the method, one study examiningthe immediate antibacterial efficacyof the bar soapcontaining1.5% TCC relativeto the vehiclebar, while the secondstudyexaminesthe prolongedantibacterialefficacyof the sametwo bars. STUDY
Twenty(20) subjects, maleandfemale,ages18 to 65 years,wereenrolledin eachstudy. Thesesubjectswere screened for normalskin on the forearms.In orderto clearthe skin of any residualantimicrobialspreviouslyused,subjectswereinstructedto useonly the non-medicated personalcleansingbar soapprovidedin placeof their regularpersonal cleansingproductduring a 7-14-day washoutperiod. They were also instructedto refrainfrom usingany antibacterialsoaps,medicatedlotionsand creams,and dandruff shampoos until the studywascompleted. All subjects providedwritten,informedconsent. TEST
Two bar soapswereexaminedin eachstudy.The antibacterialsoapcontained1.5% 3,
4, 4'-trichlorocarbanilide (TCC).The othersoapwasthe vehiclesoapbarcontaining no antimicrobialagents.
OF TEST PRODUCTS
In each study, assignmentof the test and placebosoapsto the subjectswas made accordingto a computer-generated randomization.Following the 7-14-day washout period,subjectsperformeda supervised washof their forearmsthree(3) timesper day at the laboratorywith their assignedbar of soap.Washeswere at leastone hour apart. To assess immediateresidualeffectiveness after washing,subjectsperformedsevenwashes: threewasheson days1 and 2 and onewashon day 3 of the test period.For evaluation of prolongedresidualeffectiveness, subjectsperformednine washes:threewasheson days 1, 2, and 3. Subjectswerepermitted to showerand batheduring the test period, using a non-antibacterialbar soap.However, they were instructed not to wash their arms. Showeringand bathingwasrestrictedafterthe lastwash.Subjectswerepermittedto use a non-medicated lotionon their forearmswheneverthe forearmsbecamedry, exceptafter the final wash.
At eachwashvisit, the subjectswere closelysupervisedas they washedtheir own arms. Eachforearmwaswashedseparately with the appropriatesoap.The volar surfaceof the forearmand the bar soapwere wetted under running tap water maintainedat 95ø100øF.The barwasthen rubbedon the forearmusingan up-and-downmotionfrom the wrist to the elbowfor 15 seconds. The lather wasthen rubbedon the forearmusingthe handsin the samemotion for another45 seconds, followedby a 15-secondrinse.Each forearmwaspatted dry with a paper towel.
On the lastdayof the testperiod(immediatelyafterthe seventhwashor 24 hoursafter the ninth wash), three (3) circular test sites, 3.0 cm in diameter, were marked on the
volar surfaceof eachforearm.Thesecircularsiteswere evenlyspacedalong the midsection of eachforearm,avoidingthe areaon the wrist and the elbow crease.Eachtest site was inoculatedwith S. a•reus,strain 502A (ATCC # 27217) that had been grown at 35ø + 2øC for 20 5 2 hoursin trypticasesoy broth (TSB). For inoculation,a ten-fold dilution of the S. aureusbroth culture was made, and 10 lal of the diluted bacterial culturewasappliedto eachtestsite.The inoculumconcentration rangedbetween3.4 x
105and7.8 x 105colony-forming units(CFUs)fortheinoculation immediately followingthefinalwash andbetween 1.2 x 106 and1.5 x 106 CFUsfortheinoculation 24 hoursfollowingthe final wash.Using a sterileinoculatingloop, the inoculumwas spreadwithin the centerof the test site while remaining4 to 5 mm from the marked edge.
Eachinoculated testsitewasimmediatelyoccluded with a 25-mm Hill Top Chamber © without the non-wovenabsorbentpad. The chambersweresecuredto the skin with an adhesive dressing (Durapore ©, 3M). Sitesremainedoccluded for intervalsof 30 minutes, two hours, or five hours.
At the completionof eachocclusioninterval, the chamberswere removedand the sites weresampledfor survivingorganismsusingthe cupscrubtechnique(1,2). The sampling
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solution consistedof 0.075 M phosphatebuffer with 0.1% Triton X-100 detergentat pH 7.9 and letheenas the neutralizer.Letheenhasbeenpreviouslyvalidatedand long usedin this laboratoryas a suitableneutralizerfor formulationscontainingTCC. The
samples were serially diluted inhalf-strength (0.0375 M)buffer inten-fold dilutions to 10- . Aliquots(0.1 ml) of eachundilutedanddilutedspecimenwerespreadin triplicate on the surfaceof trypticasesoy agar with 5% sheepblood (TSA/B). All plateswere incubatedaerobicallyfor 48 + 4 hoursat 35ø + 2øC.The numberof CFUs per dilution was determined.
In both studies,the survivingCFUs of bacteriafor eachsubjectwere enumeratedand thesevalueswere1og•otransformed. The meanlogloCFU countsfor boththe immediate and prolonged(24-hour)efficacystudiesare presentedin Table I. The differencein the loglo CFU countsbetweenthe antibacterialbar soapand the placeboare alsoshownin this table.
The log•o CFU countswere comparedusinga Wilcoxon signed-ranktest to estimate which of the bars(antibacterialor placebo)had the greatestantibacterialactivity. A binomial sign test wasalsousedto determineany significantdifferences betweenthe number of subjectsexperiencinga reductionin CFUs with the antibacterialbar soap relativeto the bar soapvehicle.The antibacterialsoapwassignificantly(p <• 0.05) more effectivethan the vehicle at controlling the growth of S. aureuson the skin at the 30-minute, two-hour, and five-hourocclusiontimes (Table I). Evenwhen the challenge was 24 hoursafter the final wash,the residualbenefit of the antibacterialsoapwas significantly(p <• 0.05) more effectivethan that observedimmediatelyafter use. The binomialdatafor subjectswith fewerCFUs is presentedin Table II. A significantly (p <• 0.05) greaternumberof subjectshad fewersurvivingorganismson the arm washed with the antibacterialsoapthan on the arm washedwith the placebosoapat all contact times. This pattern continuedeven 24 hoursafter the final wash.
Washing with either an antibacterialsoapor with a non-antibacterialsoapwill remove bacteriafrom the skin due to the surfactancy of the soapbaseand the mechanicalaction of the washprocedure.Still, washingis oftenincomplete,and the survivingbacteriacan readily recover.However, antibacterialsoapsare generallyformulatedto depositan antimicrobialagenton the skin that may remaineffectivefor an extendedperiodafter washing.As demonstratedin this work, this residualefficacycan inhibit the growth of organismsthat surviveon the skin or come in contactwith the skin subsequentto washing. Within two to five hours after contact with bacteria, individuals who had recentlywashedexhibited greater than 1 log differencebetweenthe antibacterialsoap and the soapvehicle.Theseresultsareconsistent with thosereportedby Finkeyeta/. (9) and Scalaet aL (10), in which they observeda residualdifferenceof approximately1.3 logsand 2.0 logs,respectively,betweenan antibacterialsoapcontaining1.5% TCC and
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a placebosoapafter repeatedwashing.Using this new modification,we were able to demonstrate significantinhibitionof bacterialgrowth after inoculationeven24 hours after washing. Sufficient residual antimicrobial agent remained on the skin after 24 hours that individualsexhibited greater than a 0.5 log differencebetweenthe antibacterialsoapand the vehiclesoaptwo to five hoursaftercontactwith bacteria.This effectwasobservedin at least80% of the subjectsat eachsamplingtime.
It is knownthat colonizationis a necessary precursorto the establishment of infection on the skin (11). An antimicrobialagent remainingon the skin can help preventthe colonizationof potential pathogenssuchas S. a•re• by inhibiting their growth and survival.SinceS. a•e•s cancolonizethe skin and causeskin infections,washingwith an antibacterialsoapwith 1.5% TCC has the potential to aid in the preventionof these diseases. As in previousstudies(8-10), TCC effectivelyinhibited the growth ofS. a•re•s in this study. The resultspresentedheresuggesta methodof greatutility to evaluatethe antimicrobial substantivityor residualefficacyof antibacterialsoapsundersimulateduseconditions. As an extensionof the methoddescribed by Aly and Maibach(8) and Finkeyetal. (9), this improvedmethodpermits the assessment of substantivityup to 24 hoursafter
washing.The Hill Top Chambers © provideconsistent,reliableconditionsthat are optimal for bacterialsurvivaland growth on the skin, contributingto reproducible results.With minimal changes,the method is amenableto evaluatingvariousproduct
formssuchasliquid soaps,showergels,andhandsanitizers or to evaluatingthe efficacy of personalcleansingproductsagainstother organismssuchas Escherichia coil Further work is neededwith productscontainingother antimicrobialagentsto establishthe utility of the methodfor evaluationof a broadrangeof products.
The authorswish to thank Dr. RazaAly, Departmentof Dermatology,Universityof California Schoolof Medicine, San Francisco,California, for his extensiveand valuable technicalguidancein the developmentof this method.
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