Comprehensive characterization of Mycobacterium tuberculosis strains after acquisition of isoniazid resistance

Includes bibliographical references.2016 Fall.Despite the global efforts to reduce tuberculosis (TB) rates, the emergence of drug resistant TB has not allowed effective control of this disease. In the last decade, there were roughly 10 million new TB cases per year and isoniazid resistant (INHr) TB accounted for 9.5% of these cases around the world. In 2012, United States had an interruption in the supply of isoniazid (INH), which increased the likelihood of INH resistance rates. Although INH resistance in Mycobacterium tuberculosis (Mtb) is multigenic, mutations in the catalase-peroxidase (katG) gene predominate amongst INHr Mtb strains. The characterization of the Mtb proteome before and after acquiring INH resistance remains understudied. Additionally, the effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. The purpose of this work is to describe a complete biochemical and immunological characterization of the INHr acquisition in Mtb. In this way, a global exploration of the protein and mycolic acids differences in Mtb cultures, as well as differences in the immune response and bacterial virulence in the mouse model comparing clonal susceptible and INHr pairs of Mtb were evaluated. After this, common trends were analyzed and the findings were interpreted in the context of bacterial metabolism and host-interaction. For this work, two clonal clinical Mtb strains and one laboratory clonal pair of the H37Rv strain with different susceptibility profiles to INH were studied. The H37Rv INHr strain was isolated from a mouse that was exposed to INH in the lab and developed the same katG mutation that one of the clinical INHr strain has (V1A). In all cases, the first strain was susceptible to all tested drugs (mostly known as the INHs strain in this dissertation) while the second strain was resistant only to INH (named INHr throughout this work). The clinical pairs were confirmed as clonal pairs of the Beijing and T genotype respectively by spoligotyping and restriction fragment polymorphism analysis that uses the patterns given by the distribution of the insertion sequence (IS)-6110. Previous whole genome sequencing analysis of the clinical clonal pairs showed a katG mutation and the presence of some additional non-synonymous polymorphisms in the INHr strains. After the proteomic analysis, a katG PCR sequencing confirmed two mutations in katG for the T INHr pair (V1A and E3V) while the L101R mutation previously identified for the Beijing INHr was not confirmed. This mutation was highly unstable and the Beijing INHr might have reversed its phenotype after the absence of INH during in vitro growth. Therefore, the analysis with the Beijing clonal pair is only presented in chapter II. Protein comparison of secreted and cellular fractions (membrane, cytosol and cell wall) between clinical and lab clonal pairs of Mtb before and after acquisition of INH resistance revealed at least 25 commonly altered proteins looking at the same cellular fractions. These proteins were involved in ATP synthase machinery, lipid metabolism, regulatory events, virulence, detoxification and adaptation processes. Western blot analysis supported some of our findings, particularly the lower level of bacterial enzyme KatG in the INHr strains. Mycolic acid (MA) analysis in these clonal pairs did not reveal a common trend in these molecules for INHr strains but generated supporting information about an alternative fatty acid biosynthetic pathway in the clinical INHr strain. These analyses are further described in chapter III. Additionally, differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of KatG were evaluated in the C57BL/6 mouse model. The results in the mouse study support the idea of the individual effect of specific located mutations in the katG gene together with the associated changes in the bacterial proteome induce differences in the Mtb virulence and pathogenicity. In addition, the in vivo results also suggest the contribution of innate immune response via TLR-2 in the clearance of the INHr-attenuated Mtb strains. Further details of this work are described in chapter IV. This work provides a better understanding of new compensatory mechanisms in Mtb after INH resistance acquisition providing novel information that could be used to address alternative combined therapies as well as the identification of new drug targets in INHr strains. The results presented here also contribute to the generation of new hypothesis regarding RNA decay in Mtb and the need to evaluate if the observed biochemical differences are also associated with the bacterial exposure to the first line drug therapy that occurred in the patient. After the results obtained in this study, a subsequent biochemical analysis of Mtb strains obtained from patients before and after drug treatment is proposed to improve the description of the evolution of the acquired drug resistant phenomena observed in TB cases that limit the global disease control and hence its eradication (chapter V)

Análise funcional do ventrículo esquerdo em angio-TC coronária

Doutoramento em Engenharia InformáticaCoronary CT angiography is widely used in clinical practice for the assessment of coronary artery disease. Several studies have shown that the same exam can also be used to assess left ventricle (LV) function. LV function is usually evaluated using just the data from end-systolic and end-diastolic phases even though coronary CT angiography (CTA) provides data concerning multiple cardiac phases, along the cardiac cycle. This unused wealth of data, mostly due to its complexity and the lack of proper tools, has still to be explored in order to assess if further insight is possible regarding regional LV functional analysis. Furthermore, different parameters can be computed to characterize LV function and while some are well known by clinicians others still need to be evaluated concerning their value in clinical scenarios. The work presented in this thesis covers two steps towards extended use of CTA data: LV segmentation and functional analysis. A new semi-automatic segmentation method is presented to obtain LV data for all cardiac phases available in a CTA exam and a 3D editing tool was designed to allow users to fine tune the segmentations. Regarding segmentation evaluation, a methodology is proposed in order to help choose the similarity metrics to be used to compare segmentations. This methodology allows the detection of redundant measures that can be discarded. The evaluation was performed with the help of three experienced radiographers yielding low intraand inter-observer variability. In order to allow exploring the segmented data, several parameters characterizing global and regional LV function are computed for the available cardiac phases. The data thus obtained is shown using a set of visualizations allowing synchronized visual exploration. The main purpose is to provide means for clinicians to explore the data and gather insight over their meaning, as well as their correlation with each other and with diagnosis outcomes. Finally, an interactive method is proposed to help clinicians assess myocardial perfusion by providing automatic assignment of lesions, detected by clinicians, to a myocardial segment. This new approach has obtained positive feedback from clinicians and is not only an improvement over their current assessment method but also an important first step towards systematic validation of automatic myocardial perfusion assessment measures.A angiografia coronária por TC (angio-TC) é prática clínica corrente para a avaliação de doença coronária. Alguns estudos mostram que é também possível utilizar o exame de angio-TC para avaliar a função do ventrículo esquerdo (VE). A função ventricular esquerda (FVE) é normalmente avaliada considerando as fases de fim de sístole e de fim de diástole, apesar de a angio-TC proporcionar dados relativos a diferentes fases distribuídas ao longo do ciclo cardíaco. Estes dados não considerados, devido à sua complexidade e à falta de ferramentas apropriadas para o efeito, têm ainda de ser explorados para que se perceba se possibilitam uma melhor compreensão da FVE. Para além disso, podem ser calculados diferentes parâmetros para caracterizar a FVE e, enquanto alguns são bem conhecidos dos médicos, outros requerem ainda uma avaliação do seu valor clínico. No âmbito de uma utilização alargada dos dados proporcionados pelos angio- TC, este trabalho apresenta contributos ao nível da segmentação do VE e da sua análise funcional. É proposto um método semi-automático para a segmentação do VE de forma a obter dados para as diferentes fases cardíacas presentes no exame de angio- TC. Foi também desenvolvida uma ferramenta de edição 3D que permite aos utilizadores a correcção das segmentações assim obtidas. Para a avaliação do método de segmentação apresentado foi proposta uma metodologia que permite a detecção de medidas de similaridade redundantes, a usar no âmbito da avaliação para comparação entre segmentações, para que tais medidas redundantes possam ser descartadas. A avaliação foi executada com a colaboração de três técnicos de radiologia experientes, tendo-se verificado uma baixa variabilidade intra- e inter-observador. De forma a permitir explorar os dados segmentados, foram calculados vários parâmetros para caracterização global e regional da FVE, para as diversas fases cardíacas disponíveis. Os resultados assim obtidos são apresentados usando um conjunto de visualizações que permitem uma exploração visual sincronizada dos mesmos. O principal objectivo é proporcionar ao médico a exploração dos resultados obtidos para os diferentes parâmetros, de modo a que este tenha uma compreensão acrescida sobre o seu significado clínico, assim como sobre a correlação existente entre diferentes parâmetros e entre estes e o diagnóstico. Finalmente, foi proposto um método interactivo para ajudar os médicos durante a avaliação da perfusão do miocárdio, que atribui automaticamente as lesões detectadas pelo médico ao respectivo segmento do miocárdio. Este novo método obteve uma boa receptividade e constitui não só uma melhoria em relação ao método tradicional mas é também um primeiro passo para a validação sistemática de medidas automáticas da perfusão do miocárdio

Feature Paper in Antibiotics for 2019

There has been much speculation about a possible antibiotic Armageddon; this would be the result of having untreatable post-operative infections, and similarly untreatable complications after chemotherapy. The now famous “O’Neill Report” (https://amr-review.org/) suggests that more people could die from resistant bacterial infections by 2050 than from cancer. We are still learning about all the subtle drivers of antibiotic resistance, and realizing that we need a single “whole of health” co-ordinated policy. We ingest what we sometimes feed to animals. There do not seem to be any new classes of antibiotics on our horizon. Perhaps something that has been around “forever” will come to our rescue—bacteriophages! Nevertheless, we have to do things differently, use antibiotics appropriately, for the correct indication, for the correct duration and with the correct dose, and with that, practice good antibiotic stewardship. Whilst by no means comprehensive, this book does cover some of the many topics of antibiotic stewardship. It also addresses some of the older antibiotics, some new combinations, and even some new agents. Last, and by no means least, there are two excellent articles on bacteriophages

SINGLE-NUCLEOTIDE RESOLUTION VIEW OF GENE EXPRESSION IN ESCHERICHIA COLI K-12 UNDER VARIOUS PHYSIOLOGICAL CONDITIONS

We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during logarithmic- growth and upon entry into stationary phase under carbon, nitrogen, and phosphate starvation conditions. To generate high-resolution transcriptome maps, we developed a quantitative method for first annotating and then calculating the three features that define an operon: the promoter, terminator, and deep RNA sequence read coverage to connect the two transcript ends. Based upon the annotation of transcription features we were able to calculate relative promoter activities, terminator efficiencies, and transcription unit activities for 2,122 promoters, 1,774 terminators, and 1,510 operons, respectively. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3’ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5’ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Additionally, we sought to identify and characterize RpoS-dependent operons, genes and promoters under carbon, phosphate and nitrogen starvation. RpoS-dependency was identified using DEseq software. Following differential expression analysis by DEseq, only transcription units, genes and promoters that were statistically significant (p-value ≤ 0.05) and demonstrated a 4-fold or greater change in expression were classified. As a result of our analysis 315 operons, 317 genes, and 278 promoters were classified as being RpoS-dependent. It was observed that RpoS-dependency was most impactful when the culture was starved for carbon, accounting for two-times more differentially regulated transcription units than nitrogen or phosphate starvation. Significant differences in the structure of RpoS-dependent transcripts were observed when compared to RpoS-independent transcripts. It was determined that most RpoS-dependent operons are monocistronic and are approximately half the size of RpoS-independent operons. Analysis of the -10 regions of the 278 putative RpoS-dependent promoters determined that the most abundant nucleotide sequence was CTACGCTTAA, a significant deviation from the consensus motif (CTATAATTAA). We hypothesize that the presence of guanine and cytosine nucleotides (CGC) at base locations -8 through -10 results in the preferential binding of RpoS to these promoter regions, whereas the vegetative sigma factor RpoD would not bind. Additionally, four new RpoS-dependent transcripts were identified within the intergenic regions of the E. coli genome. These results and conclusions describe RpoS-dependency at the operon, gene, and promoter levels, and elucidate the “core” of the RpoS regulon under three different starvation conditions

Deciphering the physiology of drug tolerant and resistant Mycobacterium tuberculosis

Thesis (PhD)--Stellenbosch University, 2021.ENGLISH ABSTRACT: Poor adherence to treatment for tuberculosis (TB) disease and the rising incidents of drug resistant Mycobacterium tuberculosis strains are factors that negatively influence TB control. The current study was designed to explore some of the key knowledge gaps concerning M. tuberculosis physiology; looking at the effect of the diverse M. tuberculosis genetic backgrounds and the presence of ropB mutation on the transcriptome, looking at M. tuberculosis host response and the likelihood as to whether induced mycobacterial tolerance can provide a reservoir from which genetic resistance can arise. Exploring some of these key knowledge gaps was imperative, given the fact that, lengthy anti-TB drug treatment could be required to entirely eradicate some of M. tuberculosis strains, and non-compliance with completing treatment might lead to the emergence of multidrug (MDR)-TB. Firstly, we investigated the effect of M. tuberculosis strains with different genetic backgrounds on their total transcriptomic profiles (as a proxy for the physiological state). Secondly, we examined the influence of rpoB Ser531Leu mutation and the effect of isoniazid (INH) treatment (24h) at sub-lethal concentrations on the transcriptomic profiles of rifampicin (RIF)-resistant (K636RIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains, using RNA-sequencing (RNA-Seq) and Real-Time quantitative polymerase chain reaction (RT-qPCR) techniques. RNA-Seq analysis identified significantly differentially expressed genes in the transcriptomes of K636WT, H37RVWT and K636RIF M. tuberculosis strains. Our comparative transcriptomic data of K636WT relative to H37RvWT M. tuberculosis strains demonstrated that different genetic backgrounds influenced the total transcriptome. We demonstrated that rpoB Ser531Leu mutation has an impact on the transcriptional responses of K636WT relative to K636RIF M. tuberculosis strains. Our data did not demonstrate an effect of INH treatment on the transcriptomes of M. tuberculosis strains from different genetic backgrounds, making this one of our limitations. We then assessed the host immune response after infection with RIF-resistant (K636RIF and H37RvRIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains using the luminex x multi-analyte profiling (xMAP) technology and enzyme-linked immunosorbent assay (ELISA). Our host response data (Chapter 4) revealed no differences in host response to K636WT and H37RvWT M. tuberculosis strains from different genetic backgrounds. In contrast, there were differences in host response to K636WT and K636RIF M. tuberculosis strains in a RAW264.7 macrophage model of infection. This was confirmed by the observed varying secretion levels of cytokines and chemokines (IL-6, IL-12p40 and RANTES) required to mediate M. tuberculosis growth and survival after 24 - 48h of infection. We further investigated whether viable but non-replicating (VBNR) persisters Mycobacterium smegmatis sub-populations, when exposed to high INH concentrations, may provide a pool from which genetic resistant mutants can arise. We used a combination of a fluorescence dilution (FD) reporter system, flow cytometry and fluorescence-activated cell sorting (FACS) to detect, quantify and separate VBNR and actively replicating (AR) M. smegmatis bacterial populations following INH treatment. Subsequently, we performed PCR to amplify the katG and inhA promoter and Sanger sequencing to identify mutations in these genes that are commonly associated with INH resistance. Our flow cytometry results showed successful detection of VBNR and AR bacterial populations in M. smegmatis::pTiGc following INH pre-treatment at high concentration (30x MIC) for 72h. Mutation frequencies of different sorted populations were determined as 3.51% for M. smegmatisVBNR, 5.20% for M. smegmatisAR and 0.02% for M. smegmatis UNT. Sanger sequencing data demonstrated a high percentage of mutations in the inhA promoter (C-15T) (76% in VBNR; 64% in AR) compared to mutations in the katG gene (48% in VBNR; 44% in AR). However, the difference was not statistically significant (p > 0.1). This study addressed the following knowledge gaps: it advanced our understanding about the M. tuberculosis physiology. It confirmed that strain genetic background and the presence of rpoB Ser531Leu mutation may play a role in the physiological state of M. tuberculosis strains as reflected in their transcriptomes. It confirmed that host response in vitro is influenced by M. tuberculosis strain genotype and that infection with K636WT, H37RvWT and H37RvRIF M. tuberculosis strains will result in the secretion of pro-inflammatory cytokines and chemokines while infection with K636RIF M. tuberculosis strain (with rpoB Ser531Leu mutation) might induce secretion of anti-inflammatory cytokines (second line of host defense). This study was the first to successfully use a FACS analysis in combination with the FD reporter system to detect, isolate and quantify VBNR from AR M. smegmatis, following INH pre-treatment at high concentrations. We speculate that our results showed that the VBNR persisters‟ sub-population is likely to provide a reservoir from which genetic resistant mutants can arise, when treated with high INH concentrations as made evident by the observed INH resistant mutants in VBNR M. smegmatis. This work contributed further knowledge to finding better strategies to prevent the spread of emerging MDR, as well as extensively drug resistant M. tuberculosis.AFRIKAANSE OPSOMMING: Verkeerdelike gebruik van behandeling van tuberkulose (TB) en die toename in die voorkoms van middel weerstandige Mycobacterium tuberculosis is faktore wat die beheer van TB negatief beinvloed. Die huidige studie is ontwerp om van die belangrikste gapings in die kennis rondom die fisiologie van M. tuberculosis te oorbrug. Die studie het gekyk na die effek van diverse M. tuberculosis genetiese agtergronde en die voorkoms van die rpoB mutasie op die transkriptoom van M. tuberculosis, die response van die gasheer op M. tuberculosis en die waarskynlikheid dat mikobakteriële toleransie „n poel verskaf waarvan genetiese weerstandigheid kan ontstaan. Die verkenning van verskeie van hierdie belangrike gapings in kennis was noodsaaklik gegewe die feit that langdurige anti-TB behandeling benodig kan word om totaal onstlae te raak van sekere M. tuberculosis stamme. Verkeerdelike gebruik en voltooing van behandeling mag lei tot die onstaan van multiweerstandige (MDR)-TB. Eerstens het ons die effek van die M. tuberculosis stamme met verskillende genetiese agtergronde op die totale transkriptoom ondersoek (as indikasie van die fisiologiese toestand). Tweedens het ons die invloed van die rpoB Ser531Leu mutasie en die effek van isoniazid (INH) behandeling (24 uur) teen sub-dodelike konsentrasies op die transkriptoom van rifampisien (RIF) weerstandige (K636RIF) en die sensitiewe (K636WT en H37RVWT) M. tuberculosis stamme ondersoek deur gebruik te maak van RNA-volgordebepaling (RNAseq) en werklike tyd, kwantitatiewe polimerase ketting reaksie (RT-qPCR). RNAseq analise het beduidende verskille in die uitdrukking van gene in die transkriptoom van K636WT, H37RVWT en K636RIF M. tuberculosis stamme getoon. Ons vergelykende transkriptoom data van K636WT en H37RVWT M. tuberculosis stamme het gedui dat verskillede genetiese agtergronde die totale transkriptoom beinvloed. Ons het gedemonstreer dat die rpoB Ser53Leu mutasie „n impak op die transkripsionele respons van K636WT het, relatief tot K636RIF M. tuberculosis stamme. „n Beperking van die studie was dat ons data nie die effek van INH behandeling op die transkriptoom van M. tuberculosis stamme van verskillende genetiese agtergronde gedemonstreer het nie. Met die assessering van die gasheer se immuun reaksie na infeksie met die RIF-weerstanding (K636RIF en H37RVRIF) en die sensitiewe (K636WTen H37RVWT) M. tuberculosis stamme deur die gebruik van luminex x multi-analiet profiel (xMAP) tegnologie en ensiem geassosieerde immunosorbens analises (ELISA) analises. Ons gasheer respons data (Hoofstuk 4) het geen verskille in gasheer reaksies tot K636WT en H37RvWT M. tuberculosis stamme van verskillende genetiese agtergronde getoon nie. In teenstelling, daar was verskillle in die gasheer respons tot K636WT en K636RIF M. tuberculosis stamme in a RAW264.7 makrofaag infeksie model. Dit is bevestig deur die waarneming van variërende sekresie vlakke van sitokienes en chemokiene (IL-6, IL-12p40 en RANTES) wat benodig word om M. tuberculosis groei en oorlewing te bemiddel 24 – 48 ure na infeksie. Ons het verder ondersoek ingestel of lewendige nie-repliserende (VBNR) persister Mycobacterium smegmatis sub-populasies, wanneer blootgestel aan hoë dosisse INH konsenstrasies, „n poel kan verskaf waarvan geneties weerstandige mutasies kan ontstaan. Ons het „n kombinasie van „n fluoresensie verdunning (FD) sisteem, vloeisitometrie en fluoreserensie-geaktiveerde sel sortering (FACS) gebruik om VBNR en aktief repliserende (AR) M. smegmatis populasies in respons tot INH behandeling te identifiseer, kwatifiseer en isoleer. Gevolglik het ons PKR gebruik om die katG en inhA promoter te amplifiseer en Sanger volgorde bepaling is gebruik om mutasies in hierdie gene te identifiseer, wat algemeen geassosieer is met INH weerstandigheid. Ons vloesitometrie resultate het gewys dat die identifisering van VBNR en AR bakeriële populasies in M. tuberculosis::pTiGc na voorafbehandeling met hoë konsentrasies INH (30x MIC) vir 72 uur. Mutasie frekwensies van verskillende gesorteerde populasies is bepaal as 3.51% vir M. smegmatis VBNR, 5.20% vir M. smegmatis AR en 0.02% vir M. smegmatis UNT. Sanger volgordebepaling het gedemonstreer dat „n hoë persentasie van die mutasies in die inhA protomoter (C-15T) (76% in VBNR; 64% in AR) geleë is, in vergelyking met mutasies in die katG geen (48% in VBNR, 44% in AR), maar die verskille was nie statisties beduidend nie (p>0.1). Hierdie studie adresseer die volgende gapings in kennis: dit bevorder ons begrip van M. tuberculosis fisiologie. Dit bevestig dat genetiese agtergrond van stamme en die voorkoms van die rpoB Ser153Leu mutasie „n rol mag speel in die fisiologiese toestand van M. tuberculosis stamme soos gereflekteer in hul transkriptoom. Dit bevestig dat die gasheer respons in vitro beinvloed word deur M. tuberculosis stam genotipes en dat die infeksie met K636WT, H37RVWT en H37RVRIF M. tuberculosis stamme sal lei to die sekresie van pro- inflammatoriese sitokienes en chemokiene, terwyl infeksie met K636RIF M. tuberculosis stamme (met die rpoB Ser531Leu mutasie) sekresie van anti-inflammatoriese sitokiene (2e linie gasheer beskerming) kan induseer. Hierdie is die eerste studie wat FACS analises in kominasie met die FD sisteem suskesvol gebruik om VBNR van AR M. smegmatis na die vooraf toediening van hoë konsentrasies INH te identifiseer, kwantifiseer en isoleer. Ons spekuleer dat ons resultate toon dat die VBNR persister sub-populasie waarskynlik „n poel verskaf waarvan geneties weerstandige mutasies kan ontstaan wanneer dit met hoë konsenstrasies INH behandel word, soos bewys deur die waarneming van die INH weerstandige mutasies in die VBNR M. smegmatis populasie. Hierdie werk dra by tot verdere kennis om verbeterede strategieë te identifiseer vir die verkoming van die verspreiding van MDR-TB en uitgebreide middel weerstandige M. tuberculosis.Doctora

Microbial Secondary Metabolites and Biotechnology

Many research teams are working to demonstrate that microorganisms can be our daily partners, due to the great diversity of biochemical transformations and molecules they are able to produce. This Special Issue highlights several facets of the production of microbial metabolites of interest. From the discovery of new strains or new bioactive molecules issued from novel environments, to the increase in their synthesis by traditional or innovative methods, different levels of biotechnological processes are addressed. Combining the new dimensions of "Omics" sciences, such as genomics, transcriptomics or metabolomics, microbial biotechnologies are opening up incredible opportunities for discovering and improving microorganisms and their production