MATERIALSANDMETHODS REFERENCES

IdentificationofSerratia marcescens GenesNeededforPolymyxin BResistanceTyrellX.Jamison,EstherOrji,andRandallH.Harris

DepartmentofBiology,ClaflinUniversityOrangeburg,SC

ABSTRACT

INTRODUCTION

ACKNOWLEDGEMENTS

Figure1. GelElectrophoresisofRestrictionDigest

DepartmentofBiology,ClaflinUniversity

FUTUREPLAN

CONCLUSION

RESULTS

Over30millionpeopleintheUnitedStateswearcontactlenses.Keratitis isaninfectionofthecorneaassociatedwiththeimpropercareofcontactlensesandknowntocauselegalblindness.Serratiamarcescens isagram-negativebacteriumknowntocausekeratitis andisresistanttotheantibioticpolymyxin B.Polymyxin Bisacycliccationicpeptidesimilartoantimicrobialpeptidesmadebythecornea.WehypothesizedthatS.marcescens genesresponsibleforpolymyxin Bresistancearealsoneededforresistancetocornealantimicrobialpeptides.Previously,weusedtransposon mutagenesistogeneratefivepolymyxin BsensitiveS.marcescensmutants.Thisprojectfocusesonidentifyingthegeneconferringresistancetopolymyxin BinoneoftheS.marcescensmutants.GenomicDNAwasisolatedfromthemutantanddigestedwiththerestrictionenzymeBamHI.TheDNAwaspurifiedandtheendsoftheDNAwereself-ligated.TheligationreactionwastransformedintoEscherichiacoli.ThisprocessconvertsthetransposonandadjacentDNAintoaplasmid.TheplasmidwasisolatedfromE.coliandtheDNAnexttothetransposon wassequenced.ThesequencewascomparedtotheGenBank databaseattheNationalCenterforBiotechnologyInformation.Sequenceanalysisindicatedthatthetransposon insertedinbetweentwogenesencodinganoxidoreductaseandaLysR typeregulator.Thesegenesarepredictedtobetranscribedinoppositedirections.Futureexperimentswillbetodetermineifthetransposon interfereswithtranscriptionofthegene.

Thereareover30millionpeopleintheUnitedStatesthatwearcontactlenses.Contactlenseshelpimprovevisionwithoutdrasticallychangingaperson’sappearance.However,thesemedicaldevicesareoftennottakencareofproperly.Between40%and90%ofthosecontactlenswearersdonotfollowtheinstructionsgiventothembytheirhealthcareproviderstoproperlytakecareoftheircontactlenses.Impropercareofcontactlensescanleadtotheformationofabiofilm onthecontactlenses.Oncethatbiofilm hasformedandthecontactlenscomesincontactwiththeeye,keratitis,aninfectionorinflammationofthecornea,canoccur.Keratitiscanhavethemajorconsequenceoflossofvisionorlegalblindnessifleftuntreated.Eachyear,bacterialresistancecontinuestobeanissueintheUnitedStatesandinhealthcarefacilitiessuchashospitalsandnursinghomes.Becausebacteriaarebecomingmoreandmoreresistant,itisbecomingincreasinglydifficulttotreatinfectionsandconditionscausedbybacteria.

Serratia marcescens isagram-negativebacteriacommonlyfoundalmostanywhereinnature.ThisbacteriaisapartofthegenusSerratia,whichhas14speciesrecognizedwithinit.Outofthese14species,8areassociatedwithhumaninfection.Serratia marcescens isthemostcommonspeciesofthegenusSerratia thatisknowntobeahumanpathogen.Serratia marcescens wouldcommonlygiveoffaredpigment,whichmadeitagoodbiologicalmarkerforinfection.Thoughthisredpigmentwaspresentinenvironmentalstrains,somenon-pigmentedisolateswerefoundtobethecauseofinfectioninhealthcarefacilities.15-30%ofallcasesofkeratitis arecausedbySerratia marcescens.

Thecornealepitheliumproducesantimicrobialpeptideswhichisanaturaldefensemechanismusedtofightoffinfection.AsSerratia marcescens continuestoevolveandactasasuperbug,theseantimicrobialpeptidesarenothavingthecapabilitiestofightoffthekeratitis thatiscaused.Notonlyaretheantimicrobialpeptidedefensesbeingbypassed,butsoarethedefensesofantibioticssuchaspolymyxin B.Polymyxin BisanantibioticwhichiscommonlyfoundinNeosporin.Onceacommontreatmentofinfectioncausedbygram-negativebacteria,theuseofpolymyxin Bslowlydwindleddownduetoconcernsaboutitstoxicitylevel.Withincreasingbacterialresistanceoccurring,polymyxins aremakingacomeback.

Usingtransposon mutagenesis,fivemutantswereidentifiedasbeingmoresusceptibletothecationicantimicrobialpeptidepolymyxin B.Nowthatsusceptiblemutantshavebeenindentified,myprojectistodeterminewhatgenewithinthegenomeofSerratia marcescens allowsittoconferresistancetopolymyxin B.

GenomicDNA BamH1 Nco1 DNAMarker

Figure2.EscherichiacoliTransformation

Figure3.GelElectrophoresisof

DigestedPlasmidClones 1.DNAMarker2.UndigestedClone13.BamH1DigestedClone14.UndigestedClone25.BamH1DigestedClone26.UndigestedClone37.BamH1DigestedClone3

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Figure4.Transposon PlacementafterSequencing

Table1.LegendofLaneComponentsinFigure3

Iamcurrentlyworkingtodeterminewhichgene,ifnotboth,weremutatedbythetransposon.

ThisisbeingdonebyisolatingtheRNAofthewildtypeandmutanttocompareexpressionofthelysR geneortheoxidoreductase genebetweenthetwobacteria.

ReverseTranscription– PCRwillbeusedastheprotocol.

Insteadofinsertingitselfinbetweenonegene,thetransposon ofmutant1Jinserteditselfbetweentwogenes:NAPHoxidoreductase andlysR familytranscriptionalregulator.

NIHRISEProgram:AwardNumberR25GM113740ResearchreportedinthispublicationwassupportedbytheNationalInstituteofGeneralMedicalSciencesoftheNationalInstitutesofHealthunderAwardNumberR25GM113740.TheconsentissolelytheresponsibilityoftheauthorsanddoesnotnecessarilyrepresenttheofficialviewsoftheNationalInstitutesofHealth.

CenterforDiseaseControlandPrevention.2015.HealthyContactLensWearandCareFactSheet.AccessedonOctober12,2016

Herra,C.andFalkiner,F.Serratia marcescens.Available:www.antimicrobe.org/b26.asp.AccessedNovember7,2016

Fernandez,L.,Alvarez-Ortega,C.,Wiegand,I.,etal.2013.CharacterizationofthePolymyxin BResistomeofPseudomonasaeruginosa.AntimicrobialAgentsandChemotherapy;57:110-119.

McNamara,R.,Van,R.,Tuchin,O.,etal. 1999.OcularSurfaceEpitheliaExpressmRNAforHumanBetaDefensin-2.AcademicPress;69:483-490.