Molecular epidemiology of tuberculosis in Tasmania and genomic characterisation of its first known multi-drug resistant case

Background:The origin and spread of tuberculosis (TB) in Tasmania and the types of strains of Mycobacteriumtuberculosis complex (MTBC) present in the population are largely unknown.Objective:The aim of this study was to perform the first genomic analysis of MTBC isolates from Tasmaniato better understand the epidemiology of TB in the state.Methods:Whole-genome sequencing was performed on cultured isolates of MTBC collected from2014–2016. Single-locus variant analysis was applied to determine the phylogeny of the isolatesand the presence of drug-resistance mutations. The genomic data were then cross-referencedagainst public health surveillance records on each of the cases.Results:We determined that 83.3% of TB cases in Tasmania from 2014–2016 occurred in non-Australianborn individuals. Two possible TB clusters were identified based on single locus variantanalysis, one from November-December 2014 (n = 2), with the second from MayAugust2015 (n = 4). We report here the first known isolate of multi-drug resistant (MDR)M. tuberculosis in Tasmania from 2016 for which we established its drug resistance mutationsand potential overseas origin. In addition, we characterised a case of M. bovis TB in aTasmanian-born person who presented in 2014, approximately 40 years after the last confirmedcase in the state’s bovids.Conclusions:TB in Tasmania is predominantly of overseas origin with genotypically-unique drug-susceptibleisolates of M. tuberculosis. However, the state also exhibits features of TB that areobserved in other jurisdictions, namely, the clustering of cases, and drug resistance. Earlydetection of TB and contact tracing, particularly of overseas-born cases, coordinated withrapid laboratory drug-susceptibility testing and molecular typing, will be essential for Tasmaniato reach the World Health Organisation’s TB eradication goals for low-incidencesettings

Transcriptome profiling defines a novel regulon modulated by the LysR-type transcriptional regulator MexT in Pseudomonas aeruginosa

The LysR-family regulator MexT modulates the expression of the MexEF-OprN efflux system in the human pathogen Pseudomonas aeruginosa. Recently, we demonstrated that MexT regulates certain virulence phenotypes, including the type-three secretion system and early attachment independent of its role in regulating MexEF-OprN. In this study, transcriptome profiling was utilized to investigate the global nature of MexT regulation in P. aeruginosa PAO1 and an isogenic mexEF mutant. Twelve genes of unknown function were highly induced by overexpressing MexT independent of MexEF-OprN. A well-conserved DNA motif was identified in the upstream regulatory region of nine of these genes and upstream of mexE. Reporter fusion analysis demonstrated that the expression of the genes was significantly induced by MexT in P. aeruginosa and a heterogenous Escherichia coli strain and that the conserved sequence was required for this induction. The conserved DNA motif was further characterized as the MexT binding site by site-directed mutagenesis and electrophoretic mobility shift assays. Genes containing this conserved regulatory sequence were identified across other Pseudomonas species, and their expression was activated by MexT. Thus, a novel regulon directly modulated by MexT, that includes but is independent of mexEF-oprN, has been identified

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans

Immunological corollary of the pulmonary mycobiome in bronchiectasis:The Cameb study

Understanding the composition and clinical importance of the fungal mycobiome was recently identified as a key topic in a “research priorities” consensus statement for bronchiectasis. Patients were recruited as part of the CAMEB study: an international multicentre cross-sectional Cohort of Asian and Matched European Bronchiectasis patients. The mycobiome was determined in 238 patients by targeted amplicon shotgun sequencing of the 18S–28S rRNA internally transcribed spacer regions ITS1 and ITS2. Specific quantitative PCR for detection of and conidial quantification for a range of airway Aspergillus species was performed. Sputum galactomannan, Aspergillus specific IgE, IgG and TARC (thymus and activation regulated chemokine) levels were measured systemically and associated to clinical outcomes. The bronchiectasis mycobiome is distinct and characterised by specific fungal genera, including Aspergillus, Cryptococcus and Clavispora. Aspergillus fumigatus (in Singapore/Kuala Lumpur) and Aspergillus terreus (in Dundee) dominated profiles, the latter associating with exacerbations. High frequencies of Aspergillus-associated disease including sensitisation and allergic bronchopulmonary aspergillosis were detected. Each revealed distinct mycobiome profiles, and associated with more severe disease, poorer pulmonary function and increased exacerbations. The pulmonary mycobiome is of clinical relevance in bronchiectasis. Screening for Aspergillus-associated disease should be considered even in apparently stable patients.MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio

<i>Neisseria</i> species as pathobionts in bronchiectasis

Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group.Published versio

Distinct 'Immuno-Allertypes' of Disease and High Frequencies of Sensitisation in Non-Cystic-Fibrosis Bronchiectasis

Rationale: Allergic sensitization is associated with poor clinical outcomes in asthma, chronic obstructive pulmonary disease, and cystic fibrosis; however, its presence, frequency, and clinical significance in non–cystic fibrosis bronchiectasis remain unclear. Objectives: To determine the frequency and geographic variability that exists in a sensitization pattern to common and specific allergens, including house dust mite and fungi, and to correlate such patterns to airway immune-inflammatory status and clinical outcomes in bronchiectasis. Methods: Patients with bronchiectasis were recruited in Asia (Singapore and Malaysia) and the United Kingdom (Scotland) (n = 238), forming the Cohort of Asian and Matched European Bronchiectasis, which matched recruited patients on age, sex, and bronchiectasis severity. Specific IgE response against a range of common allergens was determined, combined with airway immune-inflammatory status and correlated to clinical outcomes. Clinically relevant patient clusters, based on sensitization pattern and airway immune profiles (“immunoallertypes”), were determined. Measurements and Main Results: A high frequency of sensitization to multiple allergens was detected in bronchiectasis, exceeding that in a comparator cohort with allergic rhinitis (n = 149). Sensitization was associated with poor clinical outcomes, including decreased pulmonary function and more severe disease. “Sensitized bronchiectasis” was classified into two immunoallertypes: one fungal driven and proinflammatory, the other house dust mite driven and chemokine dominant, with the former demonstrating poorer clinical outcome. Conclusions: Allergic sensitization occurs at high frequency in patients with bronchiectasis recruited from different global centers. Improving endophenotyping of sensitized bronchiectasis, a clinically significant state, and a “treatable trait” permits therapeutic intervention in appropriate patients, and may allow improved stratification in future bronchiectasis research and clinical trials.Ministry of Education (MOE)Ministry of Health (MOH)National Medical Research Council (NMRC)Published versionSupported by the Singapore Ministry of Health’s National Medical Research Council under its Transition Award NMRC/TA/0048/2016 (S.H.C.) and Changi General Hospital Research grant CHF2016.03-P (T.B.L.). The work performed at NUS was supported by the Singapore Ministry of Education Academic Research Fund, SIgN, and National Medical Research Council grants N-154-000-038-001, R-154-000-404-112, R-154-000-553-112, R-154-000-565-112, R-154-000-630-112, R-154-000-A08-592, R-154-000-A27-597, SIgN-06-006, SIgN-08-020, and NMRC/1150/2008 (F.T.C.); J.D.C. is supported by the GSK/British Lung Foundation Chair of Respiratory Research

Geographic variation in the aetiology, epidemiology and microbiology of bronchiectasis

Bronchiectasis is a disease associated with chronic progressive and irreversible dilatation of the bronchi and is characterised by chronic infection and associated inflammation. The prevalence of bronchiectasis is age-related and there is some geographical variation in incidence, prevalence and clinical features. Most bronchiectasis is reported to be idiopathic however post-infectious aetiologies dominate across Asia especially secondary to tuberculosis. Most focus to date has been on the study of airway bacteria, both as colonisers and causes of exacerbations. Modern molecular technologies including next generation sequencing (NGS) have become invaluable tools to identify microorganisms directly from sputum and which are difficult to culture using traditional agar based methods. These have provided important insight into our understanding of emerging pathogens in the airways of people with bronchiectasis and the geographical differences that occur. The contribution of the lung microbiome, its ethnic variation, and subsequent roles in disease progression and response to therapy across geographic regions warrant further investigation. This review summarises the known geographical differences in the aetiology, epidemiology and microbiology of bronchiectasis. Further, we highlight the opportunities offered by emerging molecular technologies such as -omics to further dissect out important ethnic differences in the prognosis and management of bronchiectasis.NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Published versio

Microbiology and the microbiome in bronchiectasis

The microbiology in bronchiectasis has been historically defined by culture-based analysis of the airway microbiome and to date has largely focused on the detection and eradication of specific bacterial pathogens. Although central to our current understanding of disease, microbial culture alone masks the holistic complexity of the microbiome and does not account for potential microbial interactions that define specific clinical phenotypes such as frequent exacerbators. Advances in next-generation sequencing including their analytical technologies can further complement and build upon our current understanding of the microbiology and microbiome in bronchiectasis providing improved patient stratification with prognostic significance.Ministry of Health (MOH)National Medical Research Council (NMRC)This research is funded by Singapore Ministry of Health’s National Medical Research Council under its Clinician-Scientist Individual Research Grant (MOH-000141) (S.H.C.) and Clinician Scientist Award (MOH-000710) (S.H.C.). S.H.C is on advisory boards for CSL Behring and Boehringer Ingelheim and has received personal fees from AZ, all outside of the submitted work. M.M.A

The airway microbiome: present and future applications

Accelerated by developments in DNA sequencing technologies, our understanding of the respiratory microbiome is advancing at pace, providing unprecedented opportunities for clinical translation. Building on the early observations of sub-clinical micro-aspiration in healthy individuals, and initial culture independent microbiome studies in respiratory disease, recent work reveals an expansive microbial ecosystem that encompasses bacterial, fungal and viral constituents. This has led to major paradigm shifts including the potential importance of airway microbial networks in chronic respiratory disease states. As a complex organ system, with varying topology and a mucosal surface area exceeding that of the gut, the respiratory tract is recognized as a key site of host-microbe interaction. The airway experiences dynamic and continuous microbial exposures on breathing, shaped by climate and environmental surroundings, and is further influenced by sub clinical micro-aspiration of resident upper-airway microbes. The respiratory microbiome exists as an ecological gradient from upper to lower airway, interacting with host epithelia in balance between immune homeostasis and pathology. Current models posit that a balanced host-microbe interaction establishes in early life with a protective immune response that become dysregulated in respiratory disease. Characterization of microbial aberration as early indicators of deteriorating respiratory health is therefore a fundamental concept underpinning its potential clinical applications. Detecting microbial dysbiosis from otherwise ‘healthy microbiomes’ represents a potential opportunity for personalized phenotyping, stratification and therapeutic intervention. Despite such promise, this relatively nascent field has inherent challenges that need addressing as we seek to translate research gains in our understanding of the airway microbiome into tangible clinical applications for respiratory medicine

regulator MexT in Pseudomonas

Transcriptome profiling defines a novel regulon modulated by the LysR-type transcriptiona