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Development of an Antimicrobial Susceptibility Testing MethodSuitable for Performance During SpaceFlightJamesH. Jorgensen,1. JoyceA. Skweres,2 S.K. Mishra, 2 M. Letticia McElmeel,1 LouiseA. Maher,1 RossMulder, 3 Michael V. Lancaster4# and Duane L. Pierson5Department of Pathology, the University of TexasHealth ScienceCenter, SanAntonioTexas 78284,1 Krug Life Sciences,Houston, Texas,2 bioMerieux Vitek, Hazelwood,Missouri, 3 Accumed International, Westlake, Ohio, 4 NASA/Johnson SpaceCenter,Houston, stingin Microgravityauthor:James H. Jorgensen,Ph.D.Departmentof PathologyUniversityof TexasHealthScienceCenter7703 Floyd Curl DriveSan Antonio,Texas 78284-7750Telephone210-567-4088Fax ionsProgramCenterfor InfectiousCentersfor ntion

NA A .-. I-Z12973ABSTRACTVeryspacelittleflightwasis knownuponundertakentestsduringcardsthea Spacedilutionslactamaseandactionto o storageschedule(normalgravity)proposedof the cardsthecardsTM,Aandbrothprioryieldedin-flightto launchaboardfor 18-48Ground-basedcardsastronauts'MIC valuesnegativeIn someformattemperatureof inoculatedandandTheastronauts.researcha standardon the groundincubationof theaeruginosa.mediaat refrigerationby the monasthe leaureus,(beta-lactamaseanda t -(alamarBluedemonstrateddeterminedof the Vitekthe mission'sindicatorhascloacae,initiation,MICscolito thoseof inoculatedof MICsreproducibleto bacterin somein the Vitekof rchcephalosporins,endpointsstudy6-11 es,OH).testto includetetracycline,of f a malby incorporationresultswereenvironmenton bacterialpreparederythromycin,by visualsusceptibility8ocfor conductingvancomycin,International,microgravitya simpleHazelwood,determinedof theagentstrimethoprim/sulfamethoxazole.werehavethe affectsof antimicrobialof 14 antimicrobialinhibitor,drugstheregardingwilloftheh priorstudiesfor 7 daysactivities.be generatedat 4Forby

ite.Thisinformationclassesandprocedureon theof antibiotics.incubationcan provideaffectsof a seconda safeof microgravityset of testandcompactcardsin a laboratoryexperimenton the biologicalthatactivitiesatshouldof

lcraftcurrentlySpacethe effectProjectsusceptibility7 spacecraftfromof 4,6,14).SpacewasE. coliwereindicatedreturnAgencytestedto normalanalysestheanderythromycinweremore(6,14).in a staticagainstthefiveS. acoliantibioticsthatthebywereMICsthanforin ground-D1 missionto a single(microgravitywasflightthe Salyutas indicatedIn this experiment,incubatorTestin ulturesconditions)in theandin-flighttestscontrols.of microorganismsmicrobes,of . coliflight-in bothapparentis knowntheteamin vitroto increase1 g. Increasedspace-grownlevelsthecolistinsimulatedto the ground-thatsaida second(1,10,13).testedin the spaceof bacteriaduringof solatesstudieslittleantimicrobialisolatesIn a 1985 experimentdemonstrateda centrifugecomparedagainstastronautsSpaceon theincludingby a French-Sovietchloramphenicol,twiceEarly(4). Relativelyincreasedthe Russianmissionson the susceptibilityfromof the cosmonautskanamycincontrolsperformedin 1982 (3,14).to be approximatelydurationthis decade.flightbaselinewaslongeraboardon microorganisms,recoveredResistance90 dayscell densitysomewhatwereonethe flight.effectsspaceto udieseventhanduringincreasedBacteriaas comparedduringdistinctandagents.in 1975isolatesratesandare plannedof microgravityantimicrobialof tstimeshaveon thethe resultsground,of theseofinas

initial experiments, the apparent increasein resistancedid not appear to be an acquiredcharacteristic of the organisms, but rather a temporary adaptation to conditions ofmicrogravity. Electron-microscopy of space-grown cultures have shown an increaseincell-wall thickness (3). This may have altered cellular permeability, thus affecting thepenetration of antibiotics into the cells. However, increased resistanceto temporarymodificationbe excluded.Thus,affectssomeis someor classesof cumed,duringcommonthatShuttleusesMO)for manualbothtest verselysignificancein theandinoculabe handledof this studyantimicrobialpathogens.liquidsfor susceptibilitysafelywasto be conductedpreparedof ythe useprecludecannotbacterialallowwithoutto designof currentA susceptibilityincorporatinga growthsusceptibilitydeterminationsaon an cards(alamarBlue M;by astronautsmission.A ATERIALSBacterialclinicaldo notThe purposeevaluateHazelwood,OH)flightor agarwouldagainstWestlake,a eduredevelopedcouldspacein cell physiologya microgravityprolongedtestingthatrelevanceof brothsafetysimple,whichto prepareor platesor rapeuticof ontainingtests,theredrugseventualityof molecularto antibioticsEscherichiaANDof coli ,pathogensutilizedconsistingin theseA inosaof

ATCC 27853,KlebsiellaselectedbecauseAntimicrobialof knownagentsinclusionin orated30 wellin ainedampicillin,amoxicillinin a fixed1-32 sreadingstesttg/ml0.06-8(basedtestfor eachorganismVitekcardsmediatg/ml,or grouppossible,on thespecies,4-128and0.06-16trimethoprimcards(based3 and4on tedandas indicateda redoxof a2-64 tg/mlon the g/ml;0.004-0.12media.of theg/ml;negativein standardantibioticon eithertg/mlcardsmediumcephalothin0.06-162:1 ratio);ciprofloxacin,0.06-2formulatedin a fixedThefor -32 tg/ml;dilutions0.25-16For gramgrowthlag/ml;ciprofloxacin,1:19 ratio).synthesissusceptibilityVitek.of each2 ;inhibitor,researchwithouttwo1 andaction.proteingyraseby antibioticsof approximatelytest methoxazole.for this studyThea rangefor eachg/ml;cards.becauseforof antimicrobiala f theutilityand(combinedandprovidedVitekto illins,metabolismlog2 dilutionsclearagentsclavulanatethe folatewereclassesof the strainsstudyagentsclinicalchemicalSeveralto sactivecloacae.or resistanceon theirinhibitorandEnterobacterfor study.of differenttetracycline,ciprofloxacin;makebasedthe cell wallthe torbasedIn ordertoon

(alamarBlueTMAccumed,U.S. dindicatorturnedfromdefinea suitableorganism.(Difco)A lecardsStoragestandardat 35 Ccarddaysthepriorfor 16-20at 4-8 Cinoculatedto incubation.chosenof thethencards.eitherfor a definedwereIn otheror at roominoculumwithtestsuspensioninFor gram-positivesuspensionwasin 1.8 ml of testcardsdensityusingmedium.a Vitekin each50 1wellvacuumof thefilledx 105 CFU/ml.thencardsfinalandFor gram-negatives,placedto fill the VitekThe1-3andused(Becton-for the bacterialbacterialabove.resazurin).filled0.5 imentswasfinala freshwasBrothfor thesetotest mediumDetroit,simultaneouslyas thethetestto 0.0045%resazurin)weretest mediumsuspensionof the Vitekholderhourscards(in saline)manner.approximatelyor storedexperiments,usualwasto 0.003%test mediumwereincubationVitekincubatorsevenin sitiveto a 0.5 McFarlandof the McFarlandinoculatedBBL Sceptorby first preparingin turbidityspecialTMwhenfor eachLaboratories,[nowmedium.wereP. aeruginosa,(DifcoalamarBlueVitektheaccomplishedto 1.8 ml of thefillingThe100 tl of a 1:10 dilutionof a 1:10 rationbrothSparks,Biosciencesinto eachto be visuallyindicator(equivalentTMby AlamarPreliminaryE. faecalis,by ceaetriplealamarBlueof thewasMueller-HintonandFor Groupaddedandadjustedprovidedof a test organismto brighttest mediumof cationextract0.45%growthbluekindlyOhio])For the staphylococci,consistedyeastno. 5,501,959;Theinoculatedplacedperiodstoredin a standardpriorunderexperiments,temperaturewere35 Cto incubation.refrigerationVitekfor 48 hours.7cardscardsplacedin abacteriologicIn mostfor approximatelywereincubatedeither

DeterminationindividualwellsIf growthbrightof MlCsoccurredpink,antibioticspeciallyin theandthe specialexaminedindicatinglack of growthto thatwereusingvisuallypresencedesignedfor a changeto thechangedata sheetwhichof the cards,of the representedbluetoConversely,indicatedby markingtheindicator.at that test concentration.(i.e., remainingThe data wereincubationin colortheantibioticof a wellconcentration.Followingof an pinkthe configurationwellon aof thatcard.Determinationclinicalof referenceorigin,microdilutionStandardstheMICsof the studyprocedure(NCCLS)non-fastidious(7).brothwasof susceptibilityreadingsof the specialfor rminedhorseincludedresultsA Streptococcusin this isolateofto the brothbrothblood-supplementedcomparedisolatesfor ClinicalMueller-HintonThe MIC resultswerecultureaccordingCommitteeadjustedfor the Groupresults.Vitekcontrolcation3% lysedusedwereFor the stockby the edforadjusted(7).usingmanualto the NCCLS(7), or to NCCLSexpectedreferencerangesbrothon the test isolates.RESULTSUsea growthRelativelygrowthof theindicatorenriched,of theBecausereadtheresultsconfiguredVitekMICsto be iallyof theandclarityrecordedcardstest mediasharplydefinedanddeterminedweregrowthof the visualindicatoron the specialreportcard.8incorporationusedwithoutin orderend-pointssystem,sheetsof alamarBlueasdifficulty.to providewitheachTMtheVitekin less thanrapidindicatorcardcould30 secondsperbe

Preliminarystoredat 4 Cadverselyorderthestudiesfor sevenaffectingto theNCCLSdeterminedMICsby suefrommethodsat 4 Cantimicrobialtendedto be higherwhenat aThistoflight.in the specialusing35octo eightagent-organismcontrolandthefor sevenrange,Theor thatweretrimethoprim/in the rencecardsinmodule.determinedMIC values.NCCLStakento launchandMICsthe NCCLShigherpriorandwithoutwasincubatedtest cardstestprocedureapproachwouldof NCCLSoutsideh at 35 dthenof the inoculatedtherebe inoculatedon the groundinoculateby usein MICswereThisa dby storagethatthannot depicted).concernscouldfor 16-18temperature,safetydetermined1-2).to incubationusinggoodtest cardsin a rior(datathe missiondifficultieswasthe Vitekat refrigeratedinoculumTheredaysto be inoculatedduringthethattest resultsstoredtimeavoidor eightthecardsindicatedcardsthanthe dthanincubationthatthe referencethe (Tablesa preciseof the study.cefuroximethanwerevaluespreventedin this partandthatof the Vitek(e.g.,4).In someand25922).roomat 35 C (Tablestherethe o the conventionalof the nderalsocomparable3 andE. coli ATCCvaluescardsoften,wereof some35oca numberof thecardMICswithS. aureustheVitekincubation,agentswerecardof off-lowerATCCMICsas comparedwerewith

DISCUSSIONThe risk of infectious diseasesis increasedduring spaceflight becauseof the crewworking and living in crowded conditions, the useof reclaimed/recycled air and water,the absenceof infection isolation facilities, as well as human physiological changes insuch an environment (1,8,9). In addition, the observed attenuation of the humanimmune responsereported by American, Russian, and other international investigatorsmay be the most obvious reason for an increasedrisk of infectious diseasesduringspaceflight (2,5,12). The dosageof antibiotics for infections that develop in spacemaybe affected by alterations in the absorption rates and the pharmacokinetics andpharmacodynamics of antibiotics that have been demonstrated during spaceflight (11).Any decreasein microbial susceptibility to antibiotics during spaceflight couldmarkedly influence decisions on the management of infectious diseasesthat mightdevelop during long-duration missions. Severalprevious studies, although limited inscope,suggestthat bacteria cultivated in microgravity may show increased resistancetocertain antibiotics (3,4,6,10,13,14).The methodology described in this report would allow simple determinations ofthe antimicrobial susceptibility of several bacterial speciesto be made by astronautsduring a SpaceShuttle mission. The color end point method of defining MICs canprovide on-board measurementsof susceptibility during the prolonged period ofmicrogravity that occurs during a shuttle flight. The feasibility of incubating the testcards at ambient room temperature was demonstrated in this study in the event that35 Cincubation could not be provided on board the SpaceShuttle.The MICs recorded in this ground-based pilot study were reproducible andsometimes encompassedwithin the NCCLS referenceranges with four ATCC controlstrains commonly used in clinical laboratory susceptibility determinations. However,some MIC values were higher when determined in the Vitek cards. Elevated10