Investigating the relation between persister formation and clinical outcome in Tuberculosis (TB) patients

Date
2021-03
Journal Title
Journal ISSN
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Despite progressive research regarding Mycobacterium tuberculosis, Tuberculosis (TB) still remains the top cause of mortality worldwide, with South Africa being considered one of the top ten TB burdened countries. Once infected with M. tuberculosis, TB disease can progress to an active disease state, or in the majority of cases, to an asymptomatic infection state known as latency or Latent TB infection (LTBI). LTBI has been associated with recurrent TB infection after a cured TB treatment outcome was achieved as individuals with LTBI are considered reservoirs of active M. tuberculosis. A subpopulation of bacteria known as persisters is thought to contribute to the LTBI state. Persisters are viable but non-replicating (VBNR) bacteria, which are recalcitrant to antibiotic treatment. There are major knowledge gaps regarding VBNR bacteria and their role in TB treatment outcome. Previously it was observed that patients who underwent TB treatment had remaining lesion activity post- treatment and presence of M. tuberculosis mRNA suggested the presence of unculturable bacteria likely being persisters. Based on positron emission tomography – computed tomography (PET/CT) scans patients were characterized as cured, recurrent or failed. In this study, we aimed to evaluate the correlation between persister formation and pulmonary TB (PTB) disease outcome. We exploited a dual fluorescence replication reporter plasmid, and assessed persister formation using a THP-1 infection model, which mimics the host environment pathogenic mycobacteria encounter upon infection. Whole genome sequencing (WGS) data of baseline and follow-up isolates was obtained to determine if isolates are genetically predisposed to persister formation. A total of eighteen baseline clinical M. tuberculosis isolates were selected for this study. Eight isolates represented bacteria from the cured patient group while ten isolates represented bacteria from the failed/recurrent patient group. Isolates were determined to be pure cultures and WGS data was obtained. In preparation for persister assay experiments, all eighteen isolates were transformed with the fluorescence dilution (FD) dual reporter plasmid pTiGc. Growth curves demonstrated that plasmid carriage had no impact on bacterial growth. The infection model enriched for persister-like cells as reflected by a subpopulation of VBNR bacteria. We found that all bacterial isolates possessed a level of replication heterogeneity at baseline both in vitro and intracellularly. Furthermore, isolates from the cured patients showed a significantly lower frequency of persister cells compared to that of isolates from the ailed/recurrent patient group. This suggests that the inherent tendency to form persister-like cells may have an impact on PTB treatment outcome. Data suggests that persister-like cell formation may be strain dependent. However, WGS data analysis were inconclusive. Furthermore, we recognize that the sample size is a crucial limiting factor in this study and further investigation with a larger cohort would be essential. This is the first study to use clinical strains of M. tuberculosis, obtained from failed/recurrent treatment outcome group, coupled with fluorescent reporters in combination with WGS data to investigate the relationship between persister formation and clinical outcome. Possible future work would be to to validate the phenotypic study findings in a murine model. Furthermore, future studies that determine the role of genetic variation in persister formation would greatly advance a patient-specific treatment regimen that could decrease the lengthy treatment duration.
AFRIKAANSE OPSOMMING: Ten spyte van goeie vordering in Mycobacterium tuberculosis navorsing bly Tuberkulose (TB) een van die grootste oorsake van sterftes wêrelwyd met Suid-Afrika (SA) wat as een van die top tien mees geaffekteerde lande geag word. Sodra ‘n persoon geïnfekteer word met M. tuberculosis kan TB tot n aktiewe siekte toestand vorder of, soos in meeste gevalle, tot ‘n asimptomatiese infektiewe toestand ontwikkel, beter bekend as ‘n latente TB infeksie (LTBI). LTBI word geassosieer met ‘n herhalende TB infeksie nadat ‘n pasiënt genees is met behandeling omdat individue met LTBI as ‘n bron van aktiewe M. tuberculosis geag word. ‘n Subpopulasie van bakterieë bekend as persisters word as bydraende faktore van die LTBI toestand gesien. Persisters is lewendige maar nie-repliserende (VBNR) bakterieë wat anktibiotika behandeling kan weerstaan. Daar is groot gapings in ons kennis oor VBNR bakterieë, asook die rol van dié selle in die finale uitkoms van TB. Daar is voorheen waargeneem dat pasiënte wat TB behandeling ondergaan het steeds aktiewe letsels het na behandeling en die teenwoordigheid van M. tuberculosis mRNA gee aanduiding daartoe dat nie kultiveerbare bakterieë, moontlik persisters, steeds teenwoordig is. Gebaseer op resultate van positron emissie tomografie – berekende tomografie (PET/CT) skanderings is pasiënte in kategorieë genaamd genees, herhalend of misluk verdeel. n die studie beoog ons om te evalueer wat die korrelasie is tussen persister vorming en pulmonêre TB (PTB). Ons maak gebruik van ‘n dubbele fluoreserende replikasie plasmied en asseseer persister vorming in ‘n THP-1 infeksie model wat die omstandighede naboots wat M. tuberculosis teëkom in die gasheer. Heel genoom volgorde (WGS) data was versamel van oorspronklike asook opvolg isolate om vas te stel of isolate geneties meer vatbaar is vir persister vorming. In totaal is agtien kliniese M. tuberculosis isolate gekies vir die studie. Agt isolate verteenwoordig die tenvolle herstelde patiënt groep, terwyl tien isolate pasiënte die herhalende/mislukte groep verteenwoordig. Isolate was geïdentifiseer as rein kulture en WGS data was verkry. Ter voorbereiding van persister vorming eksperimente was al agtien isolate getransformeer met ‘n dubbel fluoreserende replikasie (FD) plasmied, pTiGc. Groeikurwes het gedemonstreer dat die plasmied geen effek het op bakteriële groei nie. Die infeksie model het verryk vir persister selle soos gereflekteer deur ‘n subpopulasie van VBNR bakterieë. Ons het gevind dat alle bakteriële isolate dui tot ‘n mate van heterogene replikasie, beide in vitro en intrasellulêr. Verder het die isolate van tne volle herstelde pasiënte ‘n aansienlike laer frekwensie persister selle gehad teenoor die isolate van die mislukte/herhalende groep. Dit dui daarop dat die natuurlike neiging van selle om persisters te vorm ‘n impak het op die uitkoms van TB behandeling. Data wys dat persister sel vorming spesifiek is tot kliniese isolate, alhoewel WGS data nie oortuigend was om hierdie observasie te ondersteun nie. Verder herken ons dat die klein aantal monsters ‘n belangrike beperkende faktor is in die studie en verdere ondersoek met ‘n groter monster poel noodsaaklik is. Hierdie is die eerste studie van sy soort wat gebruik maak van kliniese M. tuberculosis selle verkry van mislukte/herhalende pasiënte groepe met behulp van fluoreserende plasmiede en WGS data om die verhouding tussen persister vorming en kliniese uitkomste te bepaal. Toekomstige werk moet daarop fokus om die fenotipiese uitkomste in ‘n muis model te bevestig. Verder sal studies wat fokus op die effek van genetiese variasie en persister vorming groot vordering maak in ‘n meer pasiënt gefokusde benadering tot behandeling, wat die verlengde behandelings tydperk wat tans nodig is moontlik kan verkort.
Description
Thesis (MSc)--Stellenbosch University, 2021.
Keywords
Non-replicating (VBNR) bacteria, UCTD, Tuberculosis (TB), Mycobacterium tuberculosis, Latent virus diseases, Diseases -- Recrudescence
Citation