Evolutionary Genomics of Cystic Fibrosis and Nosocomial Pathogens of the Mycobacterium abscessus species complex

The $\textit{Mycobacterium abscessus}$ species complex (MABSC) consists of three subspecies, $\textit{M. a. abscessus}$, $\textit{M. a. massiliense}$ and $\textit{M. a. bolletii}$. All three of these subspecies are capable of causing opportunistic pulmonary and skin and soft tissue infections in immunocompromised individuals. Infections caused by the MABSC are particularly serious in people with underlying lung conditions such as Cystic Fibrosis (CF) as these organisms are highly antibiotic resistant and the currently available treatment is toxic. This has resulted in treatment of MABSC infections failing in up to 50% of cases. Given the increasing prevalence globally of infections caused by the MABSC, particularly in individuals with CF, there is increased urgency to improve the treatment available for MABSC infections. The overall aim of this project was to use genomic analyses to increase our understanding of how the MABSC has evolved to become an opportunistic pathogen, which in turn could potentially uncover promising targets for the development of novel antibiotics. Genomic analysis of the MABSC has already shown that lineages of the MABSC are capable of indirect person to person transmission and the extent to which transmission contributed to the increasing prevalence of MABSC in people with CF became evident when the MABSC global population structure was determined. This showed that 70% of the MABSC isolates from people with CF were attributed to lineages made up of densely clustered isolates, where acquisition via indirect person-to-person transmission, as opposed to from the environment, was more likely. This thesis used population genomic approaches to i) investigate the genetic factors that drove the emergence of the most prevalent MABSC lineages, ii) look for evidence of convergence after the clonal expansion of these lineages to understand how the MABSC was continuing to adapt and spread amongst people with CF and iii) to examine the within host evolution of these pathogens to uncover how these environmental organisms were adapting to the CF lung. This thesis also used whole genome sequencing to explore the largest known outbreak of MABSC infections, a post-surgical wound infection epidemic in Brazil. Through this research the emergence of the most prevalent MABSC lineages were found to be driven by increased opportunity, probably due to the increased number of people with CF surviving longer, as opposed to the acquisition of a common genetic determinant. Not enough signal was detected after the clonal expansion of the most prevalent MABSC lineages to come to strong conclusions about genetic factors driving their continuing expansion, but strong evidence of convergent evolution was detected between MABSC isolates evolving over time within the host. The MABSC was shown to be potentially using a similar central regulatory network in response to environmental cues from the phagosome to that of $\textit{M. tuberculosis}$. The investigation into the epidemic of post-surgical wound infections in Brazil showed that a single $\textit{M. a. massiliense}$ lineage was introduced into Brazil just prior to the initial outbreak and that this lineage subsequently spread, through several waves of transmission, to multiple cities in geographically distant areas of Brazil. This highlighted how the MABSC was capable of long distance transmission and emphasised the potential of the MABSC as a nosocomial pathogen capable of causing large scale outbreaks

Fighting antimicrobial resistance in mycobacteria: development of an antivirulence screening platform and a genetic methodology to confirm drug resistance phenotypes

¿RESUMENLa tuberculosis (TB) causó 10,6 millones de nuevos casos y 1,6 millones de muertes en 2021 según datos de la Organización Mundial de la Salud (OMS). Su principal agente causal es un único microorganismo infeccioso denominado Mycobacterium tuberculosis. Si bien existe una vacuna preventiva, la BCG, y hay antibióticos disponibles para el tratamiento de la TB, la protección variable que confiere la BCG contra la TB pulmonar y la aparición y propagación de cepas resistentes a antibióticos de M. tuberculosis en todo el mundo hacen que la TB sea hoy en día la primera causa de muerte por una infección bacteriana.La resistencia a los antimicrobianos es una de las 10 principales amenazas mundiales para la salud pública declaradas por la OMS, y preocupa especialmente en el caso de la TB. El uso inapropiado de los antibióticos disponibles durante décadas ha llevado al surgimiento de cepas de M. tuberculosis multirresistentes (MDR) y extremadamente resistentes a los medicamentos (XDR), en las que las opciones terapéuticas se reducen drásticamente. Para afrontar la problemática de la TB, se están desarrollando diferentes estrategias a nivel mundial: mejorar la prevención de la enfermedad con nuevas vacunas candidatas con mejores eficacias protectoras que la BCG y el desarrollo de nuevos medicamentos para tratar a los pacientes infectados con TB.El descubrimiento de nuevos antibióticos activos contra las formas sensibles y resistentes de la TB en la última década ha aumentado la cartera clínica, y 17 nuevas clases químicas de antibióticos se encuentran actualmente en ensayos clínicos de Fase I y II. Sin embargo, todavía existe una necesidad urgente de desarrollar nuevos antibióticos, pero también nuevas estrategias innovadoras para luchar contra la TB, como terapias dirigidas al huésped, basadas en fagos o en terapias antivirulencia.En esta tesis hemos desarrollado una plataforma de cribado y caracterización para el descubrimiento de nuevos fármacos con actividad inhibitoria contra el sistema PhoPR de M. tuberculosis. El sistema PhoPR es un sistema de dos componentes que regula una gran variedad de fenotipos de virulencia de M. tuberculosis y, en consecuencia, los inhibidores de este sistema podrían ser una estrategia antivirulencia con potencial contra la TB. La plataforma de cribado se ha establecido construyendo un panel de diferentes cepas indicadoras (reporteras) del sistema PhoPR M. tuberculosis. Estas cepas reporteras contienen un plásmido integrado en el que la proteína fluorescente verde está bajo el control de un promotor bien caracterizado regulado por el sistema PhoPR. Para poder tener una visión más amplia de la inhibición del sistema PhoPR, hemos construido un panel de cepas reporteras del sistema PhoPR utilizando diferentes promotores regulados por el sistema PhoPR (mcr7 y pks2) y que han sido introducidos en diferentes cepas de los linajes más extendidos de M. tuberculosis. Los ensayos secundarios desarrollados para caracterizar los posibles inhibidores del sistema PhoPR consisten en una evaluación transcriptómica de diferentes genes del regulón PhoPR (los genes mcr7, pks2, pks3, espA, espC y espD), y una evaluación proteómica de la secreción de diferentes efectores de virulencia (ESAT-6, CFP-10, EspA y EspC). Las cepas indicadoras y los ensayos secundarios se han validado utilizando mutantes isogénicos ¿phoPR y una molécula inhibidora del sistema PhoPR ya descrita en la literatura, llamada etoxzolamida (ETZ).También hemos explorado la posibilidad de utilizar una micobacteria no patógena, Mycobacterium smegmatis, como sustituta de M. tuberculosis para el descubrimiento de inhibidores del sistema PhoPR. Para hacerlo, hemos diseñado una cepa de M. smegmatis con su sistema PhoPR endógeno reemplazado por sistema heterólogo de M. tuberculosis. También hemos construido un mutante ¿phoPR como control del sistema PhoPR inactivo en M. smegmatis. Las cepas reporteras de M. smegmatis portadoras del sistema PhoPR de M. tuberculosis han mostrado resultados preliminares pero prometedores.Aparte de M. tuberculosis, existen otras micobacterias no tuberculosas (NTM) en las que preocupa la problemática de la resistencia antimicrobiana. Entre ellos, Mycobacterium abscessus destaca por su natural y fácil desarrollo de resistencia adquirida a antibióticos. La mayor accesibilidad en los últimos años a la secuenciación de genomas completos permite actualmente la identificación de múltiples polimorfismos de nucleótido único (SNP) potenciales que confieran fenotipos de resistencia a fármacos que deben confirmarse.En esta tesis, también hemos desarrollado una estrategia de ¿código de barras cromosómico¿ específica para establecer asociaciones directas de genotipo-fenotipo entre los SNP y los fenotipos de resistencia a los antibióticos en M. abscessus. Para ello, hemos utilizado la tecnología de recombinación para confirmar la relación genotipo-fenotipo de la mutación atpE D29A y la resistencia a bedaquilina. La tecnología de recombinación nos ha permitido introducir la mutación atpE D29A en la región específica del cromosoma de M. abscessus con el uso de un sustrato de intercambio alélico monocatenario (ssAES) y posteriormente recuperar clones resistentes a la bedaquilina. El uso de ssAES con mutaciones silenciosas adicionales como código de barras que rodea la mutación atpE D29A ha permitido confirmar fácilmente la integración específica de la mutación de interés en el cromosoma de M. abscessus mediate técnicas basadas en PCR. Esta estrategia de ¿código de barras¿ ha sido utilizada con éxito en dos fondos genéticos diferentes de M. abscessus, el aislado clínico M. abscessus SL541 y la cepa referencia de laboratorio ATCC19977. Además, hemos utilizado la estrategia de ¿código de barras¿ para confirmar la asociación de una segunda mutación de resistencia a bedaquilina, la mutación atpE A64P, demostrando la amplia aplicabilidad de esta metodología.SUMMARY Tuberculosis disease (TB) caused 10.6 million new cases and 1.6 million deaths in 2021 according to the data from the World Health Organization (WHO). Its main causative agent is a single infectious microorganism called Mycobacterium tuberculosis. Although there is a preventive vaccine, BCG, and there are antibiotics available for the treatment of TB, the variable protection conferred by BCG against pulmonary TB and the emergence and spread of drug-resistant M. tuberculosis strains all around the world make TB nowadays the first cause of death due to bacterial infection. Antimicrobial resistance is one of the top 10 global public health threats declared by the WHO, and it especially worries in the case of TB. The inappropriate use of antibiotics available for decades has led to the rise of multi-drug resistant and extensively drug resistant strains of M. tuberculosis, in which the therapeutic options are dramatically reduced. To overcome TB problematic there are different strategies being developed globally: improve prevention of the disease with new vaccine candidates with better protective efficacies than BCG, and development of new drugs to treat TB infected patients. The discovery of new antibiotics active against sensitive and resistant forms of TB in the last decade has increased the clinical pipeline, and 17 new chemical classes of antibiotics are currently in Phase I and II clinical trials. However, there is still an urgent need of developing new antibiotics, but also new innovative strategies to fight TB, like host directed, phage or antivirulence based therapies. In this thesis we have developed a platform for screening and characterization of new drugs with inhibitory activity against the PhoPR system of M. tuberculosis. The PhoPR system is a two component system which regulates a variety of virulence phenotypes of M. tuberculosis, and consequently, inhibitors of this system could be a potential antivirulence strategy against TB. The screening platform has been established constructing a panel of different PhoPR M. tuberculosis reporter strains. PhoPR reporter strains contain an integrative plasmid in which the green fluorescent protein is under control of a well characterized promoter regulated by the PhoPR system. In order to have a wider view of PhoPR inhibition, we constructed a panel of PhoPR reporter strains using different PhoPR regulated promoters (mcr7 and pks2) that were introduced into different strains comprising the most widespread lineages of M. tuberculosis (L2 and L4). The secondary assays developed to characterize potential PhoPR inhibitors consist of a transcriptomic evaluation of the PhoPR regulon (mcr7, pks2, pks3, espA, espC and espD genes), and a proteomic evaluation of the secretion of different virulence effectors (ESAT-6, CFP-10, EspA and EspC). The reporter strains and the secondary assays have been validated using isogenic ¿phoPR mutants and a PhoPR inhibitory molecule already described in the literature, named ethoxzolamide (ETZ). We have also explored the possibility of using a non-pathogenic mycobacterium, Mycobacterium smegmatis, as M. tuberculosis surrogate for the discovery of PhoPR inhibitors. To do so, we have engineered a M. smegmatis strain to replace its endogenous PhoPR system by the heterologous system from M. tuberculosis. We have also constructed a ¿phoPR mutant as control of inactive PhoPR system in M. smegmatis. Reporter strains constructed in the different M. smegmatis strains have demonstrated preliminary but promising results of the reporter plasmids in M. smegmatis strains carrying the PhoPR system from M. tuberculosis. Aside from M. tuberculosis, there are other non-tuberculous mycobacteria (NTM) in which antimicrobial resistance is worrying. Among them, Mycobacterium abscessus stands out because of its natural and easy development of acquired drug resistance. The growing use of Whole Genome Sequencing in the last years has allowed the identification of multiple potential single nucleotide polymorphisms (SNPs) conferring drug resistance phenotypes which need to be experimentally confirmed. In this Thesis we have also developed a targeted chromosomal barcoding strategy to establish direct genotype-phenotype associations between SNPs and antibiotic resistance phenotypes in M. abscessus. To do so, we have used the recombineering technology to confirm the genotype-phenotype relationship of an atpE D29A mutation and bedaquiline resistance. The recombineering technology allowed us to introduce the atpE D29A mutation into its specific loci in the M. abscessus chromosome with the use of a single strand allelic exchange substrate (ssAES) and to subsequently recover bedaquiline resistant clones. The use of ssAES with additional silent barcode mutations surrounding the atpE D29A mutation allowed to easily confirm the specific integration of the mutation of interest into the M. abscessus chromosome using PCR based techniques. This barcoding strategy has been successfully used in two different M. abscessus genetic backgrounds, the clinical isolate M. abscessus SL541 and the laboratory reference strain ATCC19977. Additionally, we have used the barcoding strategy to confirm the association of a second bedaquiline resistance mutation, the atpE A64P, demonstrating the broad applicability of this methodology.<br /

Mycobacterial genomics and structural bioinformatics: opportunities and challenges in drug discovery.

Of the more than 190 distinct species of Mycobacterium genus, many are economically and clinically important pathogens of humans or animals. Among those mycobacteria that infect humans, three species namely Mycobacterium tuberculosis (causative agent of tuberculosis), Mycobacterium leprae (causative agent of leprosy) and Mycobacterium abscessus (causative agent of chronic pulmonary infections) pose concern to global public health. Although antibiotics have been successfully developed to combat each of these, the emergence of drug-resistant strains is an increasing challenge for treatment and drug discovery. Here we describe the impact of the rapid expansion of genome sequencing and genome/pathway annotations that have greatly improved the progress of structure-guided drug discovery. We focus on the applications of comparative genomics, metabolomics, evolutionary bioinformatics and structural proteomics to identify potential drug targets. The opportunities and challenges for the design of drugs for M. tuberculosis, M. leprae and M. abscessus to combat resistance are discussed

Non-Tuberculosis Mycobacteria Infection and The Wider Lung Microbiota in Cystic Fibrosis

Non-tuberculosis mycobacteria (NTM) infections in people with cystic fibrosis (PWCF) pose significant challenges and can have detrimental effects on patient prognosis. This study aims to investigate the impact of NTM positivity on the background microbiome in PWCF, focusing on the culture-positive cohorts for Mycobacterium avium complex and Mycobacterium abscessus complex. Additionally, the relationship between genetic modulator status and alterations in microbial composition was examined. The study employed optimised detection methods using the Illumina MiSeq platform for NTM complexes in low-diversity samples. Furthermore, the study investigated the regulation of gene expression during CF exacerbation states and explored the effect of NTM positivity on patient factors, such as the percent predicted forced expiratory volume in one second (%FEV1). The study's findings demonstrate the optimization and improvement of NTM complex detection in low diversity samples, enabling a comprehensive evaluation of the effects of NTM positivity on the broader lung microbiota. The results indicate that the presence of NTM complexes does indeed alter the diversity and the composition of the lung microbiota in CF patients, including those taking genetic modulators. Moreover, patient factors, including NTM culture status, age, and exacerbation phases, were found to influence %FEV1. Additionally, the study observed correlations between the regulation of certain virulence factors and the patient factors: lung function, C-reactive protein levels and neutrophil counts. These findings contribute to the understanding of NTM infections in CF patients and highlight the need for further research in this area. The knowledge gained from this study has implications for improving diagnostics, treatment strategies, and patient management, ultimately aiming to enhance outcomes and prolong the lives of CF patients

The rapid detection of drug resistant mycobacteria

Mycobacterium abscessus is a multidrug resistant pathogen commonly isolated from patients with cystic fibrosis. Currently, there is no rapid diagnostic tool to detect the presence of M. abscessus. Rapid diagnosis followed by appropriate, prompt treatment remains the best curative approach to mitigate disease burden and halt transmission. A major bottle neck in developing a rapid diagnostic assay is DNA extraction. Mycobacterial cells are very difficult to lyse, the existing methods are time consuming resulting in long turnaround time to detect the pathogen. This study employed the use of microwave energy to rapidly release nucleic acids from microorganisms and test the ability to detect the released nucleic acids in a magnetic-bead-based sandwich hybridisation assay using specific DNA probes. Based on published genome sequences, probes targeting the rpoB and erm-41 genes of M. abscessus and M. smegmatis were designed. In a magnetic-bead-based sandwich hybridisation assay using these specific probes, M. abscessus and M. smegmatis were distinguished from non-specific isolates within 70 mins with a lower detection limit of 1 pg/μL. The disruptive effects of microwaves on biological structures has been attributed to the local generation of heat. The contribution, if any, of non-thermal factors is yet to be determined. To study the interaction of microwaves with cell membranes, the structure which represents the major barrier to DNA release, fluorescent microscopy was employed to examine the passage of different sized fluorescent dextran particles into bacterial and yeast cells following microwave exposure. The results show a transient membrane disruption, size dependent permeabilization of dextran particles into cells. In conclusion, a prototype hybridisation assay capable of detecting M. abscessus and M. smegmatis has been developed. The application of microwaves to cells induced membrane disruption allowing internalisation of varying sizes of fluorescent dextran particles and the release of intact DNA for detection in hybridisation assay

Cystic Fibrosis

Cystic Fibrosis - Heterogeneity and Personalized Treatment provides the latest research and clinical evidence for clinicians, scientists and researchers involved in the care of patients with cystic fibrosis (CF). This book outlines the burden of the CF microbiome, utilisation of CF registries to impact future care, the sequelae of hepatobiliary complication, the use of upcoming technologies to provide patient-centred care, and provides an overview of cystic fibrosis transmembrane regulator (CFTR) modulators. Looking after patients with CF is highly rewarding, allowing those of us to combine our dedication and problem-solving skills to create a personalized approach. This book is invaluable for those involved in the care of CF patients

Genomic Analysis of Antibiotics Resistance in Pathogens

The emergence of antibiotic-resistant pathogens currently represents a serious threat to public health and the economy. Due to antibiotic treatments in humans and veterinary medicine, prophylactic use and environmental contamination, bacteria are today more frequently exposed to unnatural doses of antibiotics and their selective effect.Antibiotic resistance can be encoded on chromosomes, plasmids, or other mobile genetic elements in bacteria. It may also result from mutations that lead to changes in the affinity of antibiotics for their targets or in the ability of antibiotics to act on bacterial growth or death. Exposure of bacteria, bacterial populations, and microbial communities to antibiotics at different concentrations shapes their genomic dynamics, as does the mobilisation and spread of resistance determinants. It is, therefore, essential to understand the dynamics and mobilisation of genes encoding antibiotic resistance, in human, animal, plant, and environmental microbiomes, through genomic and metagenomic approaches and bioinformatics analyses.This Special Issue gathers research publications on the horizontal transfer of antibiotic-resistance genes, their dissemination and epidemiology, their association with bacterial virulence, between bacterial genotypes and their phenotypes, and other related research topics

Evaluating Mycobacterium komossense JERR01 as a possible model organism for Mycobacterium tuberculosis antibiotic resistance studies

Multi-drug-resistant tuberculosis (MDR-TB) accounted for an estimated 410,000 cases of all TB infections in 2022 and presents a major challenge for global health. Studying TB is complicated due to the fastidiousness and high pathogenicity of Mycobacterium tuberculosis (Mtb) that requires specialized facilities, expensive equipment, and long experimentation phases. To overcome these challenges, various Mycobacterium species have been developed as model organisms that share some of the characteristics of Mtb but are less pathogenic and more amenable to experimentation have been proposed in the past. All these models have limitations, therefore new models for Mtb that better replicate properties of the pathogen are needed. This thesis investigates a mycobacterial strain, JERR01, that was originally identified as Mycobacterium komossense as a model organism for the development of antimicrobial resistance (AMR) towards key drugs used for the treatment of TB. Through initial phenotypic characterization of the strain that originated from a human urine sample, it was determined that this strain was fast-growing and shared similar antibiotic sensitivity profiles to Mtb for six antitubercular drugs, suggesting suitability for studying AMR development. Genomic characterisation of the strain and comparison to a type strain of M. komossense revealed that its original taxonomic designation was imprecise. Wider comparison genomic analyses provided evidence that JERR01 belonged to the Fortuitum-Vaccae clade of Mycobacterium but designated it as an unclassified mycobacterial species. Having identified homologs of genes associated with resistance to antitubercular drugs in M. JERR01, in vitro studies were conducted to see how resistance to rifampicin and isoniazid, two key drugs used in tuberculosis treatment, could evolve through in vitro experimentation. For rifampicin, similar mutational pathways to Mtb were identified in the rpoB gene, the primary target of resistance. The mutations associated with decreased sensitivity to isoniazid were almost exclusively associated with inactivating katG, which encodes the gene catalase-peroxidase responsible for activating the pro-drug isoniazid. This contrasts to the mutational pathways for isoniazid in Mtb which encompass a broader range of loci and mutations. Overall, this work has identified a mycobacterial strain that can safely and rapidly grow in the laboratory to investigate the development of resistance to antituberculosis drugs and has the potential to act as a model organism for studying AMR in Mtb