The effect of environmental conditions on growth and phenotype switching in Mycobacterium smegmatis

Apiyo, Davina-Nelson (2020-03)

Thesis (MEng)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Mycobacterium tuberculosis can exist within a host in a seemingly dormant state, in which it can tolerate antibiotic challenge. This non-heritable survival mechanism is thought to be the cause of latent tuberculosis (TB) infection. The viable but non-replicating population exhibits a phenotype known as antibiotic tolerance, with these cells being referred to as ‘persisters’. The intracellular environment of alveolar macrophages (a key habitat of M. tuberculosis bacilli) is detrimental to the survival of the bacteria, constituting antimicrobial effectors such as hypoxia, nutrient deficiency, nitrosative stress and acidic stress. Persistence arises when the bacilli can tolerate these host defence mechanisms. This study sought to investigate replication dynamics and phenotype switching of bacteria, but under in vitro environmental stresses — nutrient deficiency and acidic stress. Mycobacterium smegmatis (a non-pathogenic, fast growing Mycobacterium species) was used as a model for M. tuberculosis. To determine the response of M. smegmatis to the various growth stresses, two methods were utilized: mathematical modelling (for parameter estimation and prediction of cellular growth) and fluorescence dilution (FD — for examination of the persister population, using a dual-fluorescence replication reporter system). Results from the experimental studies indicated that the fluorescence reporter was suitable for measuring bacterial replication dynamics for up to four generations, when compared to other conventional techniques such as optical density. Under acidic conditions (pH 4.6 media), the acute decline in bacterial growth, based on the calculated mean fluorescence intensity, was apparent. Under circumstances of nutrient deficiency, results from the reporter were inconclusive, since its minimum intensity had been reached before the cells in the culture could be influenced by the stresses (from t = 16 h). As for mathematical modelling, optimization of the relevant growth parameters was done through a weighted non-linear least squares approach. Quantitative comparison of the optimized model to the validation data — by calculating the normalized root mean squared error (NRMSE) — revealed a relatively good fit for the pH. For each of the five validation experiments (with varying environmental conditions), the NRMSE of the pH was 13.40%, 12.67%, 13.96%, 5.28% and 3.38%. Based on these results, we conclude that the developed mathematical model was able to predict bacterial growth under diverse conditions, and that the reporter could accurately measure mycobacterial replication. Nonetheless, model predictability (more so for the biomass and ammonia variables) could be improved, by adding biochemical elements that influence the uptake and utilization of the substrates. It would also be beneficial to apply the model to slow-growing mycobacteria, to gauge its suitability in predicting M. tuberculosis growth. Finally, FD results under nutrient-deficient conditions could be made more conclusive by withdrawing the inducer of the far-red fluorescent protein at a later timepoint during the experiment. This makes the comparison of replication dynamics to the normal case more perceptible.

AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis kan bestaan binne ’n gasheer in ’n oënskynlike dormante toestand, waar dit bestand is teen antibiotika. Daar word gedink dat hierdie onoordraaglike oorlewingsmeganisme die oorsaak van latente tuberkulose (TB) -infeksie is. Die uitvoerbare maar nie-repliserende populasie vertoon ’n fenotipe bekend as antibiotika toleransie, waar hierdie selle na verwys word as ‘vashoudendes’. Die intrasellulêre omgewing van alveolêre makrofagos (’n sleutelhabitat van M. tuberculosis basille) is nadelig tot die oorlewing van die bakterieë, wat antimikrobiale effektors bewerk soos hipoksie, nutriënttekort, stikstofstres en suurstress. Vashoudendheid kom voor wanneer die basille hierdie gasheer se verdedigingsmeganismes kan verdra. Hierdie studie het beoog om replikasiedinamieka en fenotipe wisseling van bakterieë te ondersoek, maar onder in vitro omgewingstres — nutriënttekort en suurstres. Mycobacterium smegmatis (’n nie-patogeniese, vinnig-groeiende Mycobacterium-spesie) is gebruik as ’n model vir M. tuberculosis. Om die respons van M. smegmatis op die verskillende groei stresse te bepaal, is twee metodes gebruik: wiskundige modellering (vir parameterberaming en voorspelling van sellulêre groei) en fluoressente verdunning (FD — vir eksaminering van die vashoudendes se populasie, deur ’n tweevoudige-fluoressente replikasie rapporteerderstelsel te gebruik). Resultate van die eksperimentele studies het aangedui dat die fluoressente rapporteerder gepas was vir die afmeting van bakteriële replikasiedinamieka vir tot vier generasie, wanneer dit vergelyk word met ander konvensionele tegnieke soos optiese digtheid. Onder suur kondisies (pH 4.6 media) was die akute afneming in bakteriële groei, gebaseer op die berekende gemiddelde fluoressente intensiteit, duidelik. Onder omstandighede van nutriënttekort, was die resultate van die rapporteerder onbeslis, aangesien sy minimum intensiteit bereik is voor die selle in die kultuur beïnvloed kon word deur stres (van t = 16 h). Wat wiskundige modellering betref, is die optimering van die relevante groeiparameters gedoen deur ’n geweegde nie-liniêre kleinste kwadrate benadering. Kwantitatiewe vergelyking van die geoptimeerde model met die validasie data — deur die genormaliseerde wortel gemiddeld vierkantsfout (NRMSE) te bereken — het ’n relatiewe goeie passing vir die pH bekend gemaak. Vir elk van die vyf validasie-eksperimente (met variërende omgewingskondisies) was die NRMSE van die pH 13.40%, 12.67%, 13.96%, 5.28% en 3.38%. Gebaseer op hierdie resultate het ons tot die gevolgtrekking gekom dat die ontwikkelde wiskundige model die bakteriese groei onder ’n verskeidenheid kondisies kon voorspel, en dat die rapporteerder die Mycobacteria se replikasie akkuraat kon meet. Nietemin, modelvoorspelbaarheid (meer so vir die biomassa en ammoniaveranderlikes) kon verbeter word, deur biochemiese elemente wat die invloed van die opname en gebruik van die substrate beïnvloed, by te voeg. Dit sal ook voordelig wees om die model op stadiggroeiende mycobacteria toe te pas, om sy gepastheid te bepaal om M. tuberculosis-groei te voorspel. Laastens, FD-resultate onder nutriënttekortkondisies kan meer beslissend gemaak word deur die induseerder van die verste-rooi fluoressente proteïen by ’n later tydspunt tydens die eksperiment te onttrek. Dit maak die vergelyking van replikasiedinamieka met die normale gevallestudie meer merkbaar.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108241
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