?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F37148%2F&rft.title=Adaptation+of+mycobacterium+tuberculosis+to+biofilm+growth+is+genetically+linked+to+drug+tolerance&rft.creator=Richards%2C+Jacob&rft.description=Infections+of+Mycobacterium+tuberculosis+(MTB)+require+at+least+6+months+of+multiple+antibiotics+for+sterilization.+This+lengthy+antibiotic+regimen+is+widely+attributed+to+a+subpopulation+of+cells+that+acquire+phenotypic+tolerance+to+antibiotics.+MTB+readily+forms+pellicle+biofilms+at+the+air-media+interface+in+vitro.+These+MTB+biofilms+contain+more+phenotypically+antibiotic+tolerant+persister+cells+than+cultures+grown+suspended+in+liquid+medium+(planktonic).+Molecular+mechanisms+for+the+increase+in+persister+frequency+in+MTB+biofilms+remain+largely+unknown.+We+utilized+a+high-throughput+genomic+approach+(Tn-seq)+to+identify+genes+required+by+MTB+to+adapt+to+biofilm+growth%2C+but+not+planktonic+growth%2C+and+to+analyze+their+relationship+with+biofilm-+associated+stress+and+antibiotic+tolerance.+We+identified+multiple+classes+of+mutants+for+formation+of+MTB+pellicle+biofilms.+We+hypothesized+that+the+heterogeneous+microenvironments+of+MTB+biofilms+create+endogenous+stressors+that+allow+for+self-selection+of+a+population+enriched+for+stress+and+antibiotic+tolerant+cells.+Through+use+of+a+rifampicin+(RIF)-hypersensitive+mutant+%CE%94pstC2-A1+strain+that+forms+pellicle+biofilms+morphologically+indistinguishable+from+wild-type+(WT)%2C+we+observed+that+intrinsic+drug+tolerance+in+constituent+cells+of+biofilms+determines+the+frequency+of+persisters%3A+after+7+days+of+exposure+to+50+%C2%B5g%2FmL+RIF%2C+WT+biofilms+harbored+approximately+20-+fold+more+persisters+than+the+mutant.+These+findings+suggest+that+self-selection+of+tolerant+cells+during+biofilm+growth+significantly+promotes+persister+frequency.+Using+a+transcriptomic+analysisto+characterize+how+constituent+bacteria+within+MTB+biofilms+respond+to+environmental+cues%2C+we+demonstrate+biofilm-specific+induction+of+the+synthesis+of+isonitrile+lipopeptides+(INLP)%2C+which+seem+to+be+required+for+the+development+of+MTB+biofilm+architecture+based+on+mutant+analysis.+This+work+provides+further+insight+into+the+antibiotic+tolerant+persistence+of+MTB+biofilms%2C+and+identifies+a+biofilm-specific+biomarker+in+INLPS+for+use+in+further+investigation+of+this+phenomenon.+These+findings+provide+molecular+tools+and+potential+antibiotic+targets+for+investigation+of+MTB+persisters%2C+a+significant+public+health+obstacle+to+shortening+the+antibiotic%0D%0Atreatment+of+TB.&rft.date=2019-09-26&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F37148%2F8%2FJRichards_ETDDissertation.pdf&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F37148%2F7%2FJRichards_AppendixB.pdf&rft.identifier=++Richards%2C+Jacob++(2019)+Adaptation+of+mycobacterium+tuberculosis+to+biofilm+growth+is+genetically+linked+to+drug+tolerance.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++