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Adaptation of mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

Richards, Jacob (2019) Adaptation of mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

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, but not planktonic growth, 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 ΔpstC2-A1 strain that forms pellicle biofilms morphologically indistinguishable from wild-type (WT), we observed that intrinsic drug tolerance in constituent cells of biofilms determines the frequency of persisters: after 7 days of exposure to 50 µg/mL RIF, 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, we demonstrate biofilm-specific induction of the synthesis of isonitrile lipopeptides (INLP), 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, 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, a significant public health obstacle to shortening the antibiotic
treatment of TB.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Richards, Jacobjar91@pitt.eduJAR91
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairOjha, Anilanil.ojha@health.ny.govano7
Committee MemberGupta, Ppgupta1@pitt.edupgupta1
Committee MemberHatfull, Ggfh@pitt.eduGFH
Committee MemberMartinson, Jjmartins@pitt.eduJMARTINS
Date: 26 September 2019
Date Type: Publication
Defense Date: 26 July 2019
Approval Date: 26 September 2019
Submission Date: 22 July 2019
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 138
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Mycobacterium tuberculosis, biofilms, antibiotic tolerance, persisters, isonitrile lipopeptide
Date Deposited: 26 Sep 2019 16:41
Last Modified: 26 Sep 2019 21:02
URI: http://d-scholarship.pitt.edu/id/eprint/37148

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