Skin and Soft-Tissue Infections after Injury in the Ocean

14343_8milmed_164_3_198.pdf

MILITARYMEDICINE,164,3:198,1999

SkinandSoft-TissueInfectionsafterInjuryintheOcean:Culture

MethodsandAntibioticTherapyfor MarineBacteria

CDRKevenC.Reed, MSC USN*

LTMarkC.Crowell,MSC USNt

Isolatedorganismsfrom twocommonIndo-Pacificmarinean imals (EchinometramathaeiurchinsandAcanthasterplanci seastars)likely tocausepuncturewoundstorecreational beachcombers,divers, oroperationalmilitaryforcesduring amphibious assaultsdemonstratewhypractitionersshould considertheirfirstchoiceforpotentialantibiotictherapydif ferently fromtheirusualfavoriteantibiotics.The effects of thiosulfate-citrate-bile-sucrose (TCBS)agar, varyingsaltcon centrationsin thestandardmedia, andcomparisonof room temperatureincubationversus useof the 30°C(8BOF)incuba torare reviewed. The yield ofpathogenicmarinebacteriais increasedifTCBSagar isusedand morethanonetemperature isusedforincubation.Apotentiallysignificanthumanpatho gen,

Vibriovulnificus,appearsto beubiquitous.

Introduction

B ecauseourcommunityhospitalofapproximately1,000em ployeesis in an areawherescubadivingisextremelypop ular as arecreational activity,wewantedtobecertainthat our laboratorytechniciansknewhowto modifytheirbacteriological culturingtechniquestoisolateetiologicaerobesandfacultative anaerobesfromsepticwoundsacquiredin the oceanenviron ment.TheenvironmentinOkinawa,Japan, issubtropical,butit featuresagreat diversityoftropicalcoralreefspeciesthat arrivefromsoutheastAsiaontheKuroshiocurrent,whichflowsfrom Taiwanand thePhilippinesto southern Japan.Americans workinginOkinawacompleteapproximately100,000recre ationalscubadivesperyear.'Threecommon,potentialsources ofmarine-acquiredwoundsepsis werechosenforculturing:the white-spinedsea urchin

EchinometramathaeitypeA2,3(Fig.1),

thecrown-of-thornsstarfish/asteroid(COTS)Acanthaster planci (Fig.2),and the seawateritself.In our brief study of potentialmarinesourcesof halophilicfacultativeanaerobesand aerobicbacteria,mostisolateswereGram-negativebacteriaand grewonstandard culturemedia.

Materials and Methods

SampleCollection

Oneofthe authors

(K.C.R.)collected33 mlofseawaterin a glassscrew-topspecimenvialat 24.4 m(80feet of seawater [fsw))nearMaedaMisaki,Okinawa,Japan, onDecember21,

1996.Theoceantemperatureatdepththat daywas22°C(73°F).

This seawater sample was planted onfivestandard culture *DirectorforAncillaryServices,[LeadlngPettyOfficer,MicrobiologyDivision,and §AdvancedLaboratoryTechnician,U.S.NavalHospitalOkinawa, Okinawa,Japan. tMicrobiologyandBloodBankOfficer,NavalHospital,CampPendleton,CA92055. ThismanuscriptwasreceivedforreviewinDecember1997.Therevisedmanu scriptwasacceptedforpublicationinJune1998.

Reprint&Copyright©byAssociationof

MilitarySurgeonsofU.S.,1999.

HMIMelvaD.Castro,USN*

HM2

MaryL.Sloan,USN§

media for bacteria(includingthiosulfate-citrate-bile-sucrose [TCBS]forVibriosp.)approximately1hourlaterandculturedat roomtemperature.Awhite-spinedsea urchin, E.mathaeitype A,wascollectedatSeragaki,Okinawa,andplacedin a new Ziplocplasticbag.E.mathaeisnormalspinetipflorawasalso culturedat roomtemperaturelikethe seawatersample. OnMarch15,1997,seawaterwascollectedin a 33-mlglass screw-topvialat 8.8 m(29fsw).Then,at 17.7m(58fsw),five spineswereremovedfromonearmof a42-cm-diameterA. planciCOTSwithstainlesssteelscissorsandplacedin a glass screw-topvial(Fig.3).Usingsteriletechniquein themicrobiol ogylaboratory,thetechnicianplantedtheCOTSspinesandthe seawatersampleseparatelyon fivemediaeachforculturingat roomtemperatureandfivemediaeachinthe30°Cincubator.In April1997,additionalE.mathaeiurchin spines and seawater samples fromOnna,Okinawa,tidalpoolswerecollectedand cultured.

CuiturelldentificationTechniques

Mediawereinoculatedwithtest animalsandseawater.Test animals werelightlypressed against agar plates or into broth media.Agarplateswerestreakedforisolationandwereincu batedfor24 to 48 hours at roomtemperatureand30°C.After incubation,allcolonieswerestained using Gram'sstain. The techniciansusedstandardtestingtechniques easilyavailablein microbiologytexts.Forexample,Gram-positiveorganismswere testedusingacatalasetest,andGram-negativeorganismswere testedwithanoxidasetest andidentifiedusing theAnalytical

ProfileIndex.

Mediawerepreparedat ourinstitutionaccordingtotheman ufacturers'directions,andquality-controltesting was per formedaccordingtoNationalCommitteeforClinicalLaboratory Standardsguidelines.Mediaused forwoundcultures at this institution (MacConkey,bloodagar,hektoinenteric,andchoc olate)wereused.TCBSmediumwas also used to culture all specimens.InApril1997,additionalmedia(Salmonella/Shi gella andGram-negative)werealsoused.Mediasaltconcentra tionwasincreasedto

2.30/0forculturingoftheseawatersamples

and

E.mathaeitypeAurchinsfromOkinawa,Japan, inMarch

andApril1997.

Results

Bacterialisolatesatthedifferenttemperaturesusedarelisted in TableI. E.mathaeitypeAurchin spines had thefollowing isolatedmicroorganisms:Vibriovuln!ficus,Vibriocholerae, Pseudomonasaeruginosa,Aeromonassalmonicida,Micrococcus luteus,andPasteurellasp.SpinesoftheCOTShadthefollowing organismsisolated:Vibriovulnificus,Vibriocholerae,andPas teurellasp.

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SkinandSoft-TissueInfectionsafterInjuryin the Ocean199

TABLE I

BACTERIALISOLATESATDIFFERENTTEMPERATURES

Bacterial Isolates

Fig.1.One of theEchinometra mathaeitype Aurchinsculturedin this study.

Incubating

Temperature

Room temperature (23°C/74°Fj

Incubator

(30°C/86°F)

FromAnimal Spines

Aeromonas salmonicida

Micrococcusluteus

Pasteurella

sp.

Pseudomonasaerugmosa

Vibriovuln!ficus

Pseudomonas

sp.

Vibriocholerae

Vibriovuln!ficus

From Seawater

Micrococcus

luleus

Moraxellalacunata

Moraxella

sp.

Pseudomonassp.

Pseudomonas

aerugmosa

Micrococcussp.

Pasteurellasp.

Pseudomonassp.

Vibrio cholerae

Vibriovuln!ficus

Fig.2.The crown-of-thornsstarfish/asteroid.

Acanthasterplanci.on an Oki

nawan reef.westernPacific.

Fig.3.Spines of

Acanthasterplancistarfishthatwereculturedfor bacteria. In our study,aGram-positiveisolate,Micrococcussp.,was cultured fromtwodifferentseawatersamplesand theE. mathaeitypeAurchincollectedinDecember.Themicrococci grewbothatroomtemperatureandin the30°Cincubator.The generaMicrococcusandStaphylococcusarebothmembersofthe familyMicrococcaceae.Theyare distinctbiochemicallyand in theirantibioticsensitivities.' Whenourisolatedstrains ofVibriovulnificusweretestedfor sensitivityto 12differentantibiotics,theisolateswereresistant to ampicillin,cefazolin,cefuroxime,andpiperacillinandshowed anintermediateresponsetocefoxitin.OurV.vulnificuswas sensitivetociprofloxacinand 400mgsulfamethoxazole/80mg trimethoprim (Septra),aswellas toimipenem,gentamicin,ce fotaxime,smikacin,andsulfisoxazole. Serotypingof ourisolateswas notperformedbecause that wouldhaverequiredflyingorganismstoour contractspecialty laboratory,whichisanexpensewewouldnotexpectmostcom munityhospitallaboratoriestobearin routinecases.

Discussion

Thereare about 100million0.2-Jlmbacteriaineveryliterof seawater,at alldepthsand latitudes.

Throughfeedbackmech

anismsin themarinemicrobialfoodchain,the numberofbac teriain oceanwatereverywherevaries byless than afactorof 100.
5 Thesurfaceoflivingcoralsiscoveredbyamucoidmaterial that appears toincreaseduringstress. Thissurfacemucopo lysaccharidelayerprovidesa matrixforbacterialcolonization, allowingtheestablishmentofa"normalmicrobiota"thatmay be characteristicofa particularcoralspeciesorindicativeofhealth status.

Bothbacterialpopulationsandactivitywereshowntobe

higher in the surface mucopolysaccharidelayerthan in the surroundingwater mass."Jindal et al. reporteda shiftfrom Pseudomonassp.toVibriosp.whenthe stonystar coralMon tastreaannularislost itssymbioticzooxanthellae(microalgae) andbecame"bleached"/visiblywhitened."Thesignificanceand implicationsofthisobservedchangeinbacterialfloraofhealthy versusstressed coralsare stillbeinginvestigated.

In a studyof28 traumaticmarineinjuriesin

Hawaii,Vibrio

alginolyticuswasrecoveredfrom11%ofthe patients.'In that samestudy,themost commonsourceofthelacerationwascoral orrock,and most microorganismsrecoveredwerenormalskin flora.Mostofthewoundsseeninthat studywerenotsepticand weretreatedwithin12hoursofinjury.However,inalaterstudy of the microbiologyof three oceansamples,skin and dental scrapingsof twospeciesofsharks,firecoral(Milleporadicho tarnal.and alongspined sea urchin(Diademasetosuml,six speciesofVibriobacteriaconstitutedalmost 57%of the 67

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200
isolatesfromthosepotentialsourcesofInfection."Incidentally, some readers might find it interesting that one of the most potentnonprotein poisonsfoundinnature,tetrodotoxin,ispro ducedby Vibriojlscheri, Vibrioalginolyticus,Pseudomonassp., and

AlteromonasSp.

9

Vibrionaceae

The familyVibrionaceaeconsistsofthreegenerawhosemem bersproducemarinewoundinfections,namelyVibrio,Plesiomo nas,andAeromonas.10Thesebacteria aremotileby means of polar flagellation.Marinevibroisarehalophilic(theygrowbest when salt is added to their nutrient culture broth).Theyare natural residents ofseawater, oxidasepositive,fermentdex trose' andfacultativeanaerobicGram-negativecells.Aeromo nasisparticularlycommonwherefreshwaterisin contactwithmarinesystems. 10 The marinevibriosare thepredominantbacteriafoundin deepseawaterthat arepathogenicforhumans,but theyarealso presentinlargernumbers (togetherwithawidervarietyofbac teria)in estuarine and coastalwaters.11PreventingV.cholerae

01 in crabsmerelyrequires a10-minuteboil(somecholera

bacteria survivedan 8-minuteboil).11 Vibriovulnificuswasisolatedfromallthreespecimensin our study:urchin, starfish,andseawater.

Vibriovuln!ficus-infected

woundscanrapidlyprogressfromspreadingerythematousle sionstohemorrhagicbullae,high fever,bacteremia,andhypo tensionasaresultofsepticshock.Theincubationperiodcanbe as short as 1day(8-24hours).

10Withanoverallmortalityrate

exceeding500/0,itisimperativethatphysiciansareawareofthe cutaneousmanifestationsof

V.vuln!ficusinfection.Whendeath

occurs,it is usuallywithin48 hours ofhospitaladmission. 12 InMay1996,three adults(withco-morbidity,i.e.,diabetes, alcoholicliverdisease,andbreastcancerwithchronichepatitis C)diedinLosAngeles,California,aftereatingrawoystershar vested fromthreedifferentsitesin the GulfofMexico.13During the warmmonths,Vibriovulnificusis presentin50%ofoyster beds.From1988through

1995,theCentersforDiseaseControl

receivedreports of 302V.vuln!ficusinfectionsfromthe Gulf

Coaststates ofthe

UnitedStates."From1981through1992,

125personswithV.vuln!ficusinfectionswerereportedto the

FloridaDepartmentof HealthandRehabilitativeServices.Of this series, 35% died,and thefatalityratefromrawoyster associatedVibriovulnificussepticemiaamongpatientswithpre existing liverdisease was67%. 14

Tetracyclineis the drug of

choice.However,note the sevenantibioticsmentionedin the "Results"sectionthat ourV.vulnificusisolatesweresensitiveto. Aeromonassalmonicidawasisolatedin our study atroom temperaturefromone of the type A E.mathaeiurchins.A. salmonicida is thecausativeagentoffishfurunculosis,but no humancaseof infectioncausedbyA.salmonicidahas everbeen documented.Identificationof

Aeromonasspeciesin theclinical

laboratoryis dependenton theperformanceofanoxidasetest on all morphologicallydistinctGram-negativecoloniesthat do not resemblePseudomonasaeruginosa.15

WhichTemperatureforIncubation?

Becausemost ofthe seabedis morethan 2

milesdeepand morethan 80%ofthe marineenvironmentisperpetually cold (lessthan41°F/5°C),one mightassume that marinebacteria

MilitaryMedicine,Vol. 164,March1999

Skinand Soft-TissueInfectionsafterInjuryintheOcean shouldgenerallybe cultured in acold,hyperbaricchamber. However,ourpatientsdiveinsubtropicalwatersgenerally69to

85°F(21-29°C)and near the surface(lessthan 30mdepth),so

one wouldexpectfacultativeanaerobesthatfarebetterinwarm, oxygen-richwater.Wefoundthat the use ofboth23°Cincuba tion (roomtemperature)and the30°Cincubatorincreasedthe numberofspeciesofmarinebacteriaisolated. Mostmarinebacteriaaremotile,Gram-negativerods.16Of66 differentspeciesofbacteriaisolatedfrommarinewater,sedi ments,marineanimals,andmarine-acquired infectedwounds,

10speciesofPseudomonasand 12speciesofVibriobacteria

wereidentified.17

Roomtemperaturemightbe anappropriateincubatingtem

perature for bacteria fromacooler,moretemperateenviron ment such as the ocean near the southernCaliforniacoast. Althoughthe deserts may be hot in southernCalifornia,the ocean near the southern

Californiacoast istemperateyear

round.

FieldresearchersworkinginthekelpbedsnearLaJolla/

San

Diegofoundthemeantemperatureformorethan 5yearsat

17 m depth

(56feet)to be13.0°C(55.4°F).I8In contrast, the oceanaround

Florida,theBahamas,andOkinawa,Japan, is

considerably warmer.FromJune1993throughJuly1997,4 years ofdepth temperatures recordedin oneauthor's(K.C.R.) divelogsdemonstratedanOkinawanseatemperaturerangeof

20.5°C(69°F)in thecoldestlatewinter/earlyspringmonthsto

29°C(84°F)in the summerand earlyautumn months.

ChoosinganAntibiotic

The majorityof marine woundinfectionsareself-limiting. Evenwitha historyofexposuretoseawaterorsealife,wounds are commonlyinfectedbyStaphylococcusaureusandStrepto coccuspyogenes,19presumablyfromthetraumaticintroduction of the host's normal skin florainto thewound.Therefore,a terrestrialantibiotic effectiveagainstf3-lactamase-producing bacteriamightbeappropriate.

However,ampicillinandamoxi

cillinareineffectiveagainstpenicillinase-producingmicroor ganisms,andallstrainsofPseudomonasareresistanttoamoxi cillinandampicillin.20 Ifthe patient isimmunocompromisedor thewoundis new andmajor(full-thicknessordeeppuncture),anantibiotic effec tiveagainstmarinebacteriashouldbeprescribed.Theteethof severalspeciesofsharksfromdifferentgeographicareaswere shownto be sources ofinfectiousbacteria,includingVibrio species."Tokillmarinebacteria,thebestoutpatientantibiotics to use are ciprofloxacinin an adult doseof500 or 750mgby mouth every12hours;BactrimorSeptraDS(doublestrength) tablets every12hours;100mgofdoxycyclinetwiceaday;or500 mgoftetracyclinefourtimesa day.19OurVibriovuln!ficusisolate wasresistantto ampicillin,cefazolin,cefuroxime,andpiperacil lin.

Conclusions

Amajorpoint to berememberedby theprimaryphysician

awaitingcultureandsensitivitystudiesfroma crush injuryor puncture woundintheocean,ortreatingapatientwithmarine acquiredsepsis,ortreatinganimmunocompromisedpatient,is that theantibioticchosen should be effectiveagainstVibrio

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SkinandSoft-TissueInfectionsafterInjuryintheOcean

amoxicillinare notreasonablefirstchoiceswithseriousocean water-relatedinjuries.Tetracyclines,fluorinatedquinolones,or sulfamethoxazole-trimethoprimcompoundsare the more ap propriate frrst-linedrugs formarine-acquiredinfectionswhile sensitivityresults arepending. Salinityofthe oceansvarieswithdepth and latitude,but in theareaofourstudy,

26.5°NinthewesternPacific,theshallow

watersalinityisapproximately35ppt(3.5%).22Manyseas have sodium levelsofabout2.60/0NaCIor0.45MNa'.16Theyieldof pathogenicmarinebacteriais usuallyincreased ifTCBSagaris usedandbytheadditionof

2.30/0salineorseasalttotheculture

media.17.23 Onlya smallpercentageofthe bacterialtypesseenbymicro scopicexaminationofanenvironmentalsample,such as sea wateror soil,willgrowunderlaboratoryconditions.In the fu ture' the use of

DNAmolecularprobes thatrecognize

appropriate"signature"portionsofthebacterialvariableregions mayallowscientiststodirectlyidentifyindividualtypesofbac teria, even fromcomplexassemblagesin the naturalenviron merit."

Acknowledgments

Theauthorsthanktheunnamedreviewersfortheir helpful comments, as well as Dr. Robert van Woesik at

RyukyuUniversity and Dr. David

Larson,

cytopathologtst,for critical editorial reviews. Additionally, the authors thankDr. Ellen Denigris, Head, LaboratoryDepartment,Ms. Emi Matsudo, and Mr. KoheiHibinofor theirresearchsupport.

References

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species of seaurchins,with special reference to four species of the Indo-Pacific Echinometra.In BiologyofEchinometra,pp119-29.Edited by Yanagisawa, Ya sumasu,Oguro, et al. Rotterdam, TheNetherlands,Balkema, 1991.

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88-97.Edited by Williamson JA,FennerPJ,BurnettJW, et

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and Florida.

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JJ:Bacteriologyofthe teeth froma great whiteshark: potentialmedicalimplicationsfor sharkbite victims. J Clin Microbiol 1984: 20:

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LetterstotheEditor

Subject:ClinicalTraumaTrainingforHospitalCorpsman

Date:Wed,20Jan 1999 19:34:53EST

From:RBGUS@aol.com

To:milmed@amsus.org

It was withgreatinterestthat I readMajorBen-Abraham,etaI's article"PrimaryTraumaCare Expereince ofArmyReserve

CombatMedics:Is a newapproachneeded?"

(Miltary

Medicine,1999;Vol164(1)48-50).

In July, 1993NavalReserveHospitalCorpsmanfrom NR Naval HospitalCampPendletonDetachment519beganflex drilling in theEmergencyandTraumaDepartment(Level

IIat that time,

now

Levell),UniversityMedicalCenterofSouthernNevada.

Thisprogramwasconceivedandimplementedvia aMemoradum ofUnderstanding(MOU)toprovidethe"handson"experience suggestedin thearticle.

Iwouldbe more thanhappytoprovide

interestedindividualswitha summaryoftheprogram implementationand a "go by" MOU.

Respectfully,

HMC(FMF)RichardB.Gustavson

rbgus@aol.com

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