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Biomass smoke exposure is associated with a heightened risk of development of respiratory diseases that include chronic obstructive pulmonary disease (COPD). The aim of this study was to increase our understanding of how biomass smoke could contribute to an increased susceptibility to respiratory infection. We investigated the effects of cow dung and wood smoke exposure on human bronchial epithelial cells with respect to adherence of a major respiratory bacterial pathogen in COPD, nontypeable Haemophilus influenzae (NTHi), using immunofluorescence microscopy. In addition, expression of a known receptor of NTHi, platelet-activating factor receptor (PAFR), and two pro-inflammatory cytokines, interleukin 6 (IL-6) and interleukin-8 (IL-8), were determined using quantitative polymerase chain reaction. We observed a dose-dependent increase in NTHi adhesion to human bronchial epithelial cells following exposure to cow dung but not wood smoke extracts. Pre-treatment with PAFR antagonists, WEB-2086 and its analogue, C17, decreased adherence by NTHi to airway epithelial cells exposed to cow dung smoke. Both cow dung and wood smoke-induced expression of PAFR, as well as of IL-6 and IL-8, which was inhibited by WEB-2086 and C17. In conclusion, biomass smoke from combustion of cow dung and wood-induced expression of PAFR and airway inflammatory markers in human bronchial epithelial cells. Cow dung exposure, but not wood smoke exposure, mediated a measurable increase in NTHi adhesion to airway epithelial cells that was inhibited by PAFR antagonists. This work highlights the potential of PAFR as a therapeutic target for reducing the impact of hazardous biomass smoke exposure on respiratory health.

COPD is a seriously disabling respiratory condition that inexorably progresses to disability and mortality. It affects approximately 10% of the population globally with a greater prevalence at advanced ages. Airway bacterial infections complicate the disease course in most COPD patients, leading to increased symptoms, more rapid decline in lung function, acute exacerbations and reduced quality of life. With increasing bacterial resistance to antibiotics and adverse effects of conventional treatments, new effective non-antibiotic antimicrobial therapies are urgently needed to manage COPD. Hypoxia-inducible factor (HIF)-1α is an important transcriptional regulator of cellular responses to hypoxia, oxidants and inflammation, and is overexpressed in the lungs of COPD patients. Recent evidence shows that increased HIF-1α expression can upregulate the platelet-activating factor receptor (PAFR) on the airway epithelial surface that is increased in smokers and particularly COPD patients. The receptor is utilized by PAFR-dependent bacteria (Streptococcus pneumoniae, Haemophilus influenzae and Pseudomonas aeruginosa) to induce infection in both the respiratory and gastrointestinal (GI) tracts. However, the importance and mechanism of HIF-1α in augmenting PAFR-dependent bacterial infections in COPD are poorly understood. Here, we review the evidence for the roles of local tissue hypoxia-induced inflammation, HIF-1α and PAFR in facilitating bacterial infections in COPD. Blocking PAFR may provide a novel antimicrobial approach to manage bacterial infections in COPD.

The aim of this study was to assess the association between regional tidal volume (Vt), regional functional residual capacity (FRC), and the expression of genes linked with ventilator-induced lung injury. Two groups of BALB/c mice (n = 8 per group) were ventilated for 2 hours using a protective or injurious ventilation strategy, with free-breathing mice used as control animals. Regional Vt and FRC of the ventilated mice was determined by analysis of high-resolution four-dimensional computed tomographic images taken at baseline and after 2 hours of ventilation and corrected for the volume of the region (i.e., specific [s]Vt and specific [s]FRC). RNA concentrations of 21 genes in 10 different lung regions were quantified using a quantitative PCR array. sFRC at baseline varied regionally, independent of ventilation strategy, whereas sVt varied regionally depending on ventilation strategy. The expression of IL-6 (P = 0.04), Ccl2 (P

The treatment of Mycobacterium tuberculosis infection is often viewed in isolation from other human microbial symbionts. Understandably, the clinical priority is eliminating active or latent tuberculosis (TB) in patients. With the increasing resolution of molecular biology technologies, it is becoming apparent that antibiotic treatment can perturb the homeostasis of the host microbiome. For example, dysbiosis of the gut microbiota has been associated with an increased risk of the development of asthma, obesity and diabetes. Therefore, fundamental questions include: Does TB chemotherapy cause disruption of the human microbiome and adverse effects in patients; and are there signature taxa of dysbiosis following TB treatment. In this review, we examine recent research on the detection of changes in the microbiome during antibiotic administration and discuss specific findings that relate to the impact of anti-tubercular chemotherapy.

Background A wide range of bacterial infections occur in coronavirus disease 2019 (COVID-19) patients, particularly in those with severe coronaviral disease. Some of these are community-acquired co-infections. Objective To review recent data that indicate the occurrence of hospital-onset bacterial infections, including with antibiotic-resistant isolates, in COVID-19 patients. Sources Using PubMed, the literature was searched using terms including: ‘COVID-19’; ‘SARS-CoV-2’; ‘bacterial infection’; ‘healthcare-associated infection’; ‘antibiotic resistance’; ‘antimicrobial resistance’; ‘multi-drug resistance’; ‘Streptococcus’; ‘Staphylococcus’; ‘Pseudomonas’; ‘Escherichia’; ‘Klebsiella’; ‘Enterococcus’; ‘Acinetobacter’; ‘Haemophilus’; ‘MRSA’; ‘VRE’; ‘ESBL’; ‘NDM-CRE’; ‘CR-Ab’; ‘VRSA’; ‘MDR’. Content There is a growing number of reports of bacterial infections acquired by patients with severe COVID-19 after hospital admission. Antibiotic-resistant pathogens found to cause healthcare-associated infections (HAIs) in COVID-19 patients include methicillin-resistant Staphylococcus aureus, New Delhi metallo-β-lactamase-producing carbapenem-resistant Enterobacterales, carbapenem-resistant Acinetobacter baumannii, extended-spectrum β-lactamase Klebsiella pneumoniae and vancomycin-resistant enterococci. COVID-19 has impacted bacterial HAIs in a number of ways with an increase in the incidence of New Delhi metallo-β-lactamase-producing carbapenem-resistant Enterobacterales and carbapenem-resistant A. baumannii reported at some hospital sites compared with before the pandemic. Recommended guidelines for antimicrobial stewardship in COVID-19 patient treatment are discussed regarding minimization of empiric broad-spectrum antibiotic use. Other studies have reported a decrease in methicillin-resistant S. aureus and vancomycin-resistant enterococci cases, which has been attributed to enhanced infection prevention and control practices introduced to minimize intra-hospital spread of COVID-19. Implications Poorer outcomes have been observed in hospitalized COVID-19 patients with an antibiotic-resistant infection. Although heightened IPC measures have been accompanied by a reduction in some HAIs at specific sites, in other situations, COVID-19 has been associated with an increase in bacterial HAI incidence. Further research is needed to define the cost–benefit relationship of maintaining COVID-19-related infection prevention and control protocols beyond the pandemic to reduce the burden of HAIs. In addition, the longer-term impact of high usage of certain broad-spectrum antibiotics during the COVID-19 pandemic requires evaluation.

The genus Haemophilus contains a number of species of medical importance. In particular, Haemophilus influenzae causes a range of invasive diseases including meningitis, septicaemia, epiglottis, cellulitis and arthritis, as well as non-invasive infections that can present as sinusitis, otitis media, chronic bronchitis and pneumonia. Conventional laboratory biotyping and genotyping lack the resolution required to distinguish between isolates of across these different clinical phenotypes. However, advancements in whole-genome sequencing have made it possible to detect subtle differences in the bacterium’s genome content. In this chapter, we explore the potential that genomics and bioinformatics offer to the management of H. influenzae infection. In particular, we discuss specific examples of their application in the study of COPD-related infections caused by non-typeable strains of H. influenzae.

Nepal exhibits a tuberculosis (TB) incidence rate that is comparable to neighbouring high TB incidence countries. In addition, it records500 cases of multi-drug resistant (MDR) TB each year. The objective of this study was to perform whole-genome bioinformatic analysis on MDR-TB isolates from Nepal (n = 19) to identify the specific mutations underlying their phenotypic resistance. In addition, we examined the dominant genotype among the Nepal MDR-TB isolates, the East-Asian Beijing sub-lineage, to determine its relatedness to a panel of 1274 genomes of international strains available from public databases. These analyses provided evidence that the XDR-TB isolates in our collection were not derived from importation of primary XDR-TB to Nepal but were more likely the result of acquisition of second-line drug resistance in Nepal. Resistance to fluoroquinolones was detected among a high proportion of the Nepal isolates. This has implications for the management of TB, including appropriate antimicrobial stewardship and susceptibility testing for fluoroquinolones and other second-line TB drugs, to minimise the development of XDR-TB among Nepal TB cases.

The critical importance of primary health care in maintaining a healthy population is well established internationally. Nevertheless, general practitioner care is not always easily accessible for some patients in Australia, particularly in rural regions. This is partly due to an insufficient number of medical graduates entering and being retained in the rural general practitioner workforce. Key elements of international and national programs designed to address this shortfall are discussed and include the use of entry requirements that preferentially select for applicants from a rural residence background, and immersion of medical students for a large share, or entire duration, of their training in rural communities. In addition, other factors that can influence decisions to enter and stay in rural practice are discussed.

Nontypeable Haemophilus influenzae (NTHi) colonizes human upper respiratory airways and plays a key role in the course and pathogenesis of acute exacerbations of chronic obstructive pulmonary disease (COPD). Currently, it is not possible to distinguish COPD isolates of NTHi from other clinical isolates of NTHi using conventional genotyping methods. Here, we analysed the core and accessory genome of 568 NTHi isolates, including 40 newly sequenced isolates, to look for genetic distinctions between NTHi isolates from COPD with respect to other illnesses, including otitis media, meningitis and pneumonia. Phylogenies based on polymorphic sites in the core-genome did not show discrimination between NTHi strains collected from different clinical phenotypes. However, pan-genome-wide association studies identified 79 unique NTHi accessory genes that were significantly associated with COPD. Furthermore, many of the COPD-related NTHi genes have known or predicted roles in virulence, transmembrane transport of metal ions and nutrients, cellular respiration and maintenance of redox homeostasis. This indicates that specific genes may be required by NTHi for its survival or virulence in the COPD lung. These results advance our understanding of the pathogenesis of NTHi infection in COPD lungs.

Nontypeable Haemophilus influenzae (NTHi) is an important cause of human illness, including pneumonia and acute exacerbations of chronic obstructive pulmonary disease (COPD). We report here the draft genome of an isolate of NTHi collected from the sputum of a patient presenting with COPD in Tasmania, Australia.

Extensively drug-resistant (XDR) Mycobacterium tuberculosis has become a challenge to the treatment of tuberculosis (TB) in several countries, including Nepal. Here, we report for the first time the draft genome sequence of an isolate of XDR-TB collected in Nepal and describe single-nucleotide variations associated with its extensively drug-resistant phenotype.

Globally, the burden of chronic respiratory diseases (CRDs) is increasing rapidly. These include asthma, chronic respiratory obstructive diseases (COPD) and cystic fibrosis (CF). Patients with CRDs often exhibit increased levels of oxidant burden in the lungs that is primarily due to chronic exposure to deleterious particles, including cigarette smoke, air pollution, occupational exposure to chemicals and fumes and a variety of allergens. In homeostasis, a delicate balance exists between the pro-oxidant and antioxidant molecules/entities. Both structural and immune cells, when encountering these foreign particles, generally respond by triggering pro-oxidative stress-related pathways in the lungs, thereby disturbing the pulmonary redox homeostasis. Moreover, patients with CRDs are also susceptible to frequent/recurrent microbial infections that lead to worsening of disease which often requires hospitalizations. Several pathogens, such as Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Mycobacterium tuberculosis, Aspergillus fumigatus, etc., have the ability to elicit pro-oxidant pathways in the respiratory tract. Also, these pathogens are equipped with enzymatic and non-enzymatic mechanisms to neutralize host-associated oxidative molecules that facilitate the persistence of these pathogens in the lungs. We will discuss the CRD/pathogen-triggered oxidative stress in the lungs. We will also discuss the microbial mechanisms that may further increase oxidative stress in patients with CRDs that potentially results in the heightened inflammatory response in the lungs. Finally, we will discuss the current treatment strategies to limit the oxidative response-associated lung pathologies.

Globally, chronic respiratory diseases (CRDs), both communicable and noncommunicable, are among the leading causes of mortality, morbidity, economic and societal burden, and disability-adjusted life years (DALYs). CRDs affect multiple components of respiratory system, including the airways, parenchyma, and pulmonary vasculature. Although noncommunicable respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), cystic fibrosis (CF), and lung cancer (LC), account for enormous disease burden, the currently available therapies only focus on alleviating the symptoms of diseases rather than providing optimal treatment and/or prevention. Similarly a major respiratory communicable disease, that is, tuberculosis (TB), is associated with the challenge of increasingly developing antibiotic resistance in the bacterial pathogen Mycobacterium tuberculosis. In light of these challenges, we aim to summarize the underlying molecular and cellular mechanisms that lead to hallmark pathophysiology of CRDs. Moreover, we will also highlight the limitations of current therapeutic strategies and explore novel drug delivery options that may be potentially more effective in the management of CRDs.

Tuberculosis (TB), caused by the bacteriumMycobacterium tuberculosis, is the leading cause of mortality worldwide due to a single infectious agent. The pathogen spreads primarily via aerosols and especially infects the alveolar macrophages in the lungs. The lung has evolved various biological mechanisms, including oxidative stress (OS) responses, to counteract TB infection.M. tuberculosisinfection triggers the generation of reactive oxygen species by host phagocytic cells (primarily macrophages). The development of resistance to commonly prescribed antibiotics poses a challenge to treat TB; this commonly manifests as multidrug resistant tuberculosis (MDR-TB). OS and antioxidant defense mechanisms play key roles during TB infection and treatment. For instance, several established first-/second-line antitubercle antibiotics are administered in an inactive form and subsequently transformed into their active form by components of the OS responses of both host (nitric oxide,S-oxidation) and pathogen (catalase/peroxidase enzyme, EthA). Additionally,M. tuberculosishas developed mechanisms to survive high OS burden in the host, including the increased bacterial NADH/NAD+ratio and enhanced intracellular survival (Eis) protein, peroxiredoxin, superoxide dismutases, and catalases. Here, we review the interplay between lung OS and its effects on both activation of antitubercle antibiotics and the strategies employed byM. tuberculosisthat are essential for survival of both drug-susceptible and drug-resistant bacterial subtypes. We then outline potential new therapies that are based on combining standard antitubercular antibiotics with adjuvant agents that could limit the ability ofM. tuberculosisto counter the host’s OS response.

Mycobacterium tuberculosis (Mtb) is a globally distributed bacterial pathogen whose population structure has largely been shaped by the activities of its obligate human host. Oceania was the last major global region to be reached by Europeans and is the last region for which the dispersal and evolution of Mtb remains largely unexplored. Here, we investigated the evolutionary history of the Euro-American L4.4 sublineage and its dispersal to the South Pacific. Using a phylodynamics approach and a dataset of 236 global Mtb L4.4 genomes we have traced the origins and dispersal of L4.4 strains to New Zealand. These strains are predominantly found in indigenous Māori and Pacific people and we identify a clade of European, likely French, origin that is prevalent in indigenous populations in both New Zealand and Canada. Molecular dating suggests the expansion of European trade networks in the early 19th century drove the dispersal of this clade to the South Pacific. We also identify historical and social factors within the region that have contributed to the local spread and expansion of these strains, including recent Pacific migrations to New Zealand and the rapid urbanization of Māori in the 20th century. Our results offer new insight into the expansion and dispersal of Mtb in the South Pacific and provide a striking example of the role of historical European migrations in the global dispersal of Mtb.

Platelet-activating factor (PAF) and its receptor (PAFr) have been implicated in a wide range of diseases and disorders that originate from the activation of inflammatory pathways. Although the exact structure of the binding site on the PAFr remains unknown, the PAFr is a well-established therapeutic target, and an array of structurally diverse PAFr antagonists have been identified. These include compounds that are structurally similar to the natural PAF ligand, synthetic heterocycles, complex polycyclic natural products, and various metal complexes. This review provides an update on more than 20 years of progress in this area. The development and synthesis of new PAFr antagonists, structure-activity relationship studies, the biological activity of these molecules, and their therapeutic potential are discussed.

Recurrence is a characteristic feature of Nontypeable Haemophilus influenzae (NTHi) infection, particularly during COPD exacerbations, which makes management difficult. Recurrent infections are caused either by relapse from persistence of earlier infection or by new infection with an exogenous strain. Whole genome sequencing provides the ultimate resolution to differentiate between strains from new and existing infections. However, the lack of a simple workflow for genome-wide pairwise comparison between the paired isolates still represents a barrier to its adoption. We collected 5 pairs of longitudinal isolates of NTHi from sputum sampling at baseline and recurrence and sequenced their whole genome using the Illumina MiSeq platform. A workflow was created in Galaxy, a web-based scientific analysis platform to compare the number and location of single nucleotide polymorphisms (SNPs) between the isolates collected at baseline and subsequent re-infection. Low-quality bases and adaptors were removed by use of TRIMMOMATIC. The genome of the baseline isolate was assembled de novo and was annotated automatically by use of SPADES and PROKKA, respectively. We then mapped the sequence reads of re-infection isolates to the annotated reference baseline genome and variants were called using SNIPPY. Here, we identified little genetic distance, 0-27 variants, between each pair of longitudinal isolates, suggesting relapse from persistence of earlier infection. This simple workflow for analysis of whole genome sequences enables robust differentiation of relapse and new infection with high resolution, allows assessment of treatment efficacy, and provides insights into the biology of recurrence.

From 2015 onwards, the number of vancomycin-resistant Enterococcus faecium (VREfm) isolates increased in Tasmania. Previously, we examined the transmission of VREfm at the Royal Hobart Hospital (RHH). In this study, we performed a state-wide analysis of VREfm from Tasmania’s four public acute hospitals. Whole-genome analysis was performed on 331 isolates collected from screening and clinical specimens of VREfm. In silico multi-locus sequence typing (MLST) was used to determine the relative abundance of broad sequence types (ST) across the state. Core genome MLST (cgMLST) was then applied to identify potential clades within the ST groupings followed by single-nucleotide polymorphic (SNP) analysis. This work revealed that differences in VREfm profiles are evident between the state’s two largest hospitals with the dominant vanA types being ST80 at the RHH and ST1421 at Launceston General Hospital (LGH). A higher number of VREfm cases were recorded at LGH (n = 54 clinical, n = 122 colonization) compared to the RHH (n = 14 clinical, n = 67 colonization) during the same time period, 2014–2016. Eleven of the clinical isolates from LGH were vanA and belonged to ST1421 (n = 8), ST1489 (n = 1), ST233 (n = 1), and ST80 (n = 1) whereas none of the clinical isolates from the RHH were vanA. For the recently described ST1421, cgMLST established the presence of individual clusters within this sequence type that were common to more than one hospital and that included isolates with a low amount of SNP variance (≤16 SNPs). A spatio-temporal analysis revealed that VREfm vanA ST1421 was first detected at the RHH in 2014 and an isolate belonging to the same cgMLST cluster was later collected at LGH in 2016. Inclusion of isolates from two smaller hospitals, the North West Regional Hospital (NRH) and the Mersey Community Hospital (MCH) found that ST1421 was present in both of these institutions in 2017. These findings illustrate the spread of a recently described sequence type of VREfm, ST1421, to multiple hospitals in an Australian state within a relatively short time span.

Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms of costs. However, the lack of a beginner’s protocol for WGS still represents a barrier to its adoption in some settings. Here, we present detailed step-by-step methods for obtaining WGS data from a range of different bacteria (Gram-positive, Gram-negative, and acid-fast) using the Illumina platform. Modifications have been performed with respect to DNA extraction and library normalization to maximize the output from the laboratory consumables invested. The protocol represents a simplified and reproducible method for producing high quality sequencing data. The key advantages of this protocol include simplicity of the protocol for users with no prior genome sequencing experience and reproducibility of the protocol across a wide range of bacteria.

In 2016, there were an estimated 490 000 cases globally of multidrug- resistant (MDR) tuberculosis exhibiting resistance to isoniazid and rifampicin.1 The first case of MDR tuberculosis diagnosed in Tasmania occurred in 2016 in a Vietnamese- born person. Vietnam was the second highest reported country of birth for overseas- born patients with tuberculosis notified in Australia in 2014.2 The patient had previously tested positive for tuberculosis infection in an interferon- γ release assay test performed in Tasmania in early 2016, but at the time, the patient was asymptomatic and had a normal chest x- ray and a negative sputum culture. After an episode of colitis, a colon tissue biopsy specimen isolated Mycobacterium tuberculosis. Whole genome sequence of the isolate (TASMDR1), identified high confidence mutations for isoniazid, rifampicin, ethambutol and pyrazinamide, in accordance with the culture- based drug susceptibility testing, and, in addition, it identified a mutation associated with streptomycin resistance.

The burden of tuberculosis in New Zealand peaked during the Second World War with 2603 cases recorded in 1943. Control measures legislated for in the Tuberculosis Act of 1948 played a significant role in bringing down the national incidence rate from over 150 cases per 100,000 to below 10 per 100,000 by the 1990s. Today, New Zealand is considered to be a low TB incidence country; however, this designation can mask disparities in the burden of TB within the population. The Asian ethnic group exhibits rates of TB that are approximately 50 times greater than in the European or other ethnic groups. Furthermore, among New Zealand-born individuals, Māori experience a higher burden of TB compared to their Pākehā compatriots. In this chapter, factors involved in contributing to the risk of TB in New Zealand are examined. Data from molecular typing studies are explored to gain an insight into the transmission of the illness in the population. In terms of further measures to reduce the incidence of TB in New Zealand, newer technologies that specifically detect latent TB infection are required for pre-immigration screening from high TB burden countries. Greater utilisation of prophylactic therapy of latent TB infection in individuals at risk of developing TB could assist in preventing reactivated cases. And early detection and treatment of active TB cases combined with enhanced contact tracing would help minimise the emergence of epidemiological clusters of the disease.

Tuberculosis (TB) does not occur in isolation from other human illnesses. There are multiple examples where TB combines with one of more comorbidities to amplify its prevalence. Noncommunicable diseases such as diabetes, or lifestyle behaviors including smoking and alcohol misuse, place people at a greater risk of presenting with active TB. But the epidemiological associations between TB and other human conditions are not confined to increasing susceptibility to TB disease. TB, in itself, is an underlying risk factor for the development of downstream respiratory illnesses later in life. This indicates that injury to the host resulted from an episode of TB persists beyond successful eradication of Mycobacterium tuberculosis infection by antimicrobial drug therapy. In this chapter, the specific role of TB in promoting other lung diseases is examined. In particular, TB during childhood increases the risk of development of progressive and poorly reversible airway diseases that include bronchiectasis and chronic obstructive pulmonary disease. It is apparent from the literature that prevention of TB disease offers a potential pathway for reducing the global burden of downstream chronic lung diseases.

Background: COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenza (NTHi) and Streptococcus pneumonia adhere to PAFr on the luminal surface of human respiratory tract epithelial cells. Objective: To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumonia. Methods: Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumonia labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken. Results: PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumonia to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure. Conclusion: WEB-2086 represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD.

Chronic obstructive pulmonary disease (COPD) is one of the main causes of human mortalities globally after heart disease and stroke. There is increasing evidence of an aetiological association between COPD and pneumonia, the leading infectious cause of death globally in children under 5 years. In this review, we discuss the known risk factors of COPD that are also shared with pneumonia including smoking, air pollution, age and immune suppression. We review how lung pathology linked to a previous history of pneumonia may heighten susceptibility to the development of COPD in later life. Furthermore, we examine how specific aspects of COPD immunology could contribute to the manifestation of pneumonia. Based on the available evidence, a convergent relationship is becoming apparent with respect to the pathogenesis of COPD and pneumonia. This has implications for the management of both diseases, and the development of new interventions.

Chronic obstructive pulmonary disease (COPD) is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. There is growing evidence of a co-morbidity between COPD and tuberculosis (TB), the leading cause of death globally due to respiratory infection. Thus, the increase in the burden of COPD over the coming decades, as predicted by the World Health Organisation, is of concern with respect to the control of TB. A better understanding of the interactions between these two diseases is essential for the design of complementary preventive and control strategies. In this review, some of the known risk factors that are common to both diseases are discussed. Furthermore, we examine how impairment of the innate immune system, and corticosteroid therapy, in COPD patients may increase the risk of TB manifestation. Conversely, we review how TB lung pathology may heighten susceptibility to subsequent development of COPD, even after completion of effective TB treatment. Growing evidence appears to point towards a bi-directional relationship between these two lung diseases where each may act as an independent risk factor for the other. This has important implications for the respective long-term management of TB and COPD.

Neisseria meningitidis is the causative agent of cerebrospinal meningitis and that of a rapidly progressing fatal septic shock known as purpura fulminans. Meningococcemia is characterized by bacterial adhesion to human endothelial cells of the microvessels. Host specificity has hampered studies on the role of blood vessels colonization in N. meningitidis associated pathogenesis. In this work, using a humanized model of SCID mice allowing the study of bacterial adhesion to human cells in an in vivo context we demonstrate that meningococcal colonization of human blood vessels is a prerequisite to the establishment of sepsis and lethality. To identify the molecular pathways involved in bacterial virulence, we performed transposon insertion site sequencing (Tn-seq) in vivo. Our results demonstrate that 36% of the genes that are important for growth in the blood of mice are dispensable when bacteria colonize human blood vessels, suggesting that human endothelial cells lining the blood vessels are feeding niches for N. meningitidis in vivo. Altogether, our work proposes a new paradigm for meningococcal virulence in which colonization of blood vessels is associated with metabolic adaptation and sustained bacteremia responsible for sepsis and subsequent lethality.

The spread of vancomycin-resistant Enterococcus faecium (VREfm) has become a challenge to health care infection control worldwide. In 2015, a marked increase in VREfm isolation was detected in acute public hospitals in Tasmania. We report here the draft whole-genome sequence of a newly designated VREfm sequence type, sequence type 1421 (ST1421).

In 2015, a marked increase in vancomycin-resistant Enterococcus faecium (VREfm) isolation was detected at the Royal Hobart Hospital, Australia. The primary objective of this work was to examine the dynamics of VREfm transmission using whole genome data mapped to public health surveillance information. Screening and clinical isolates of VREfm from patients were typed for the specific vancomycin-resistance locus present. Of total isolates collected from 2014–2016 (n = 222), 15.3% and 84.7% harboured either the vanA or the vanB vancomycin-resistance locus, respectively. Whole-genome sequencing of 80 isolates was performed in conjunction with single-nucleotide polymorphic (SNP) analysis and in silico multi-locus sequence typing (MLST). Among the isolates sequenced, 5 phylogenetic clades were identified. The largest vanB clade belonged to MLST sequence type ST796 and contained clinical isolates from VREfm infections that clustered closely with isolates from colonised patients. Correlation of VREfm genotypes with spatio-temporal patient movements detected potential points of transmission within the hospital. ST80 emerged as the major vanA sequence type for which the most likely index case of a patient cluster was ascertained from SNP analyses. This work has identified the dominant clones associated with increased VREfm prevalence in a healthcare setting, and their likely direction of transmission.

Whole Genome Sequencing (WGS) is emerging as a very powerful tool for the management, outbreak analyses, surveillance and determining drug resistance of human infectious pathogens including Mycobacterium tuberculosis and MRSA. WGS can also discriminate relapse TB from re-infection and the resolution provided by WGS has no comparison to conventional technologies. With current cost coming down to

SUMMARYThe health status of the Irish Traveller ethnic minority is low compared to the general population in Ireland in terms of infant mortality rates and life expectancies. Respiratory disease is an area of health disparity manifested as excess mortalities in Traveller males and females. In this study, we examined the available data with regard to tuberculosis (TB) notifications in Ireland from 2002 to 2013. We found an increase in TB notifications in Irish Travellers from 2010 onwards. This resulted in a crude incidence rate for TB in Irish Travellers that was approximately threefold higher than that of the white Irish-born population in 2011 and 2012. An outbreak of TB in Irish Travellers in 2013 increased this differential further, but when outbreak-linked cases were excluded, a higher incidence rate was still observed in Irish Travellers relative to the general population and to white Irish-born. The mean age of a TB patient was 26 years in Irish Travellers compared to 43 years in the general population, and 49 years in white Irish-born. Based on available data, Irish Travellers exhibit a higher incidence rate and younger age distribution of TB compared to white Irish-born and the general population. These observations emphasize the importance of routine use of ethnicity identifiers in the management of TB and other notifiable communicable illnesses in Ireland. They also have implications for the orientation of preventive services to address health disparities in Irish Travellers and other ethnic minority groups.

The spread of multidrug-resistant (MDR) tuberculosis (TB) has become a major global challenge. In 2016, Tasmania recorded its first known incidence of MDR-TB. Here, we report the draft whole-genome sequence of the Mycobacterium tuberculosis isolate from this case, TASMDR1, and describe single-nucleotide polymorphisms associated with its drug resistance.

The spread of multidrug-resistant (MDR) tuberculosis (TB) has become a major global challenge. In 2016, Tasmania recorded its first known incidence of MDR-TB. Here, we report the draft whole-genome sequence of the Mycobacterium tuberculosis isolate from this case, TASMDR1, and describe single-nucleotide polymorphisms associated with its drug resistance.

The genome sequence of Mycobacterium tuberculosis strain H37Rv is an important and valuable reference point in the study of M. tuberculosis phylogeny, molecular epidemiology, and drug-resistance mutations. However, it is becoming apparent that use of H37Rv as a sole reference genome in analysing clinical isolates presents some limitations to fully investigating M. tuberculosis virulence. Here, we examine the presence of single locus variants and the absence of entire genes in H37Rv with respect to strains that are responsible for cases and outbreaks of tuberculosis. We discuss how these polymorphisms may affect phenotypic properties of H37Rv including pathogenicity. Based on our observations and those of other researchers, we propose that use of a single reference genome, H37Rv, is not sufficient for the detection and characterisation of M. tuberculosis virulence-related loci. We recommend incorporation of genome sequences of other reference strains, in particular, direct clinical isolates, in such analyses in addition to H37Rv.

The Rangipo strain of Mycobacterium tuberculosis achieved notoriety in New Zealand due to its role in several tuberculosis (TB) outbreaks. Why this strain should be the source of relatively large clusters of the disease is unknown. In this work, we performed an in-depth analysis of the genome of the Rangipo strain to determine whether it offers clues to understanding its prevalence.Next-generation sequencing was performed on nine isolates which matched the Rangipo genotypic profile. Sequence reads were assembled against the H37Rv reference genome and single-locus variants identified. Unmapped reads were compared against the genome sequences of other M. tuberculosis strains, in particular CDC1551, Haarlem and Erdman.Across the nine Rangipo strains, a total of 727 single-locus variants were identified with respect to H37Rv, of which 700 were common to all Rangipo strains sequenced. Within the common variants, 386 were non-synonymous, with 12 occurring in genes associated with M. tuberculosis virulence. Next-generation and Sanger sequencing determined the presence of three genes in the Rangipo isolates, which are absent in H37Rv, but which have been reported to be important for the pathogenicity of M. tuberculosis. The differentially encoded Rangipo genes consisted of transcriptional regulator EmbR2, and molybdopterin cofactor biosynthesis proteins A and B. The Rangipo strain also harbours an extended DNA helicase and an additional adenylate cyclase.Our study provides new insights into the genomic content of the New Zealand Rangipo strain of M. tuberculosis and highlights the presence of additional virulence-related loci not found in H37Rv.

Host surface receptors provide bacteria with a foothold from which to attach, colonize and, in some cases, invade tissue and elicit human disease. In this review, we discuss several key host receptors and cognate adhesins that function in bacterial pathogenesis. In particular, we examine the elevated expression of host surface receptors such as CEACAM-1, CEACAM-6, ICAM-1 and PAFR in response to specific stimuli. We explore how upregulated receptors, in turn, expose the host to a range of bacterial infections in the respiratory tract. It is apparent that exploitation of receptor induction for bacterial adherence is not unique to one body system, but is also observed in the central nervous, gastrointestinal and urogenital systems. Prokaryotic pathogens which utilize this mechanism for their infectivity include Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. A number of approaches have been used, in both in vitro and in vivo experimental models, to inhibit bacterial attachment to temporally expressed host receptors. Some of these novel strategies may advance future targeted interventions for the prevention and treatment of bacterial disease.

Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms of costs. However, the lack of a beginner’s protocol for WGS still represents a barrier to its adoption in some settings. Here, we present detailed step-by-step methods for obtaining WGS data from a range of different bacteria (Gram-positive, Gram-negative, and acid-fast) using the Illumina platform. Modifications have been performed with respect to DNA extraction and library normalization to maximize the output from the laboratory consumables invested. The protocol represents a simplified and reproducible method for producing high quality sequencing data. The key advantages of this protocol include: simplicity of the protocol for users with no prior genome sequencing experience and reproducibility of the protocol across a wide range of bacteria.

Background COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells. Objective To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae. Methods Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken. Results PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure. Conclusion WEB-2086 represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD.

Summary The level of immigration from high tuberculosis (TB) burden countries (HBCs) which impacts on the foreign-born TB notification rate is largely unknown. In this work, we performed a cross-sectional analysis of epidemiological data from 2000 to 2013 from nine European countries: Austria, Denmark, Finland, Hungary, Netherlands, Norway, Spain, Sweden, and the United Kingdom. Crude notification rates were calculated for foreign- and native-born populations and a multiple-linear regression model predicting notification rates with HBC population data was generated. From 2000 to 2013, the population percentage with a foreign birthplace increased on average each year in all nine countries, ranging from +0.11%/year in the Netherlands to +0.66%/year in Spain. An annual increase in HBC migrants above +0.43% per year (95% Confidence Interval: 0.24%–0.63%) corresponded with higher TB notification rates in the foreign-born population of the countries analyzed. This indicates that migration from HBCs can exert a measurable effect on the foreign-born TB notification rate. However, an increase in the foreign-born TB notification rate coincided with an average annual rise in national TB notification rates only in countries, Norway (+3.85%/year) and Sweden (+2.64%/year), which have a high proportion (>80%) of TB cases that are foreign-born.

Pneumonia is the single largest infectious cause of death in children worldwide and a major cause of mortality in the elderly. Tuberculosis (TB) is the leading cause of mortality due to respiratory infection worldwide, killing approximately 1.5 million people each year. It is perhaps not surprising that the bulk of applied research on these high burden diseases has focussed on the development of new vaccines, diagnostic tools, and therapeutic interventions. However, in recent years, it has become apparent that both transmissible diseases contribute to the development of a major non-communicable disease, namely, chronic obstructive pulmonary disease (COPD). The latter chronic illness is emerging as the third largest cause of human mortality worldwide after heart disease and stroke and hence, there is acute interest in understanding its genesis and development. In this review, we examine the evidence that previous lower respiratory tract infectious disease is a contributory factor in the development of COPD. Based on the available data, there is an apparent epidemiological association between pneumonia, TB and COPD in later life. In addition, elements of COPD treatment place patients at a higher risk of presenting with pneumonia or TB. There is now a need to generate a deeper understanding of the interactions between bacterial lung disease and COPD from which new, complementary preventive and co-management strategies can be designed.

Every year, an area of sea ice forms around the continent of Antarctica that is inhabited by communities of microbes. Although the microbial component of this habitat has been postulated to play an ecologically important role in the Southern Ocean, our understanding of the diversity of Antarctic sea-ice bacteria is limited. The aim of this study was to identify bacterial components present in sea ice at Cape Hallett. Total community DNA was isolated from sea-ice cores and the 16S ribosomal RNA genes were amplified by polymerase chain reaction (PCR) using a range of universal and targeted primer sets. PCR products were de-replicated using restriction fragment length polymorphism (RFLP) and distinct RFLP types were sequenced. The major bacterial classes identified consisted of α-proteobacteria, γ-proteobacteria, Cytophaga–Flavobacteria–Bacteroides group and a small number of Gram-positive species. Our findings indicate that the bacterial component of sea ice at Cape Hallett is primarily heterotrophic. Cyano...

Mycobacterium tuberculosis kills more people than any other bacterial pathogen. New drugs are required to shorten the treatment time and provide a viable therapy for drug-resistant and latent forms of tuberculosis. The tuberculosis field has advanced considerably since the publication of the M. tuberculosis genome sequence. Today, researchers can build a high definition map of the pathogen's traits and behavior and select individual targets for chemical disruption.This review examines the discovery of current clinical and candidate tuberculosis drugs. It outlines recent developments in the selection of molecular targets for the discovery of new anti-mycobacterial agents. It appraises techniques that incorporate target knowledge into the screening protocol. These techniques include in silico, in vitro enzyme-based, differential antisense sensitivity and gene expression screening systems. The review also looks ahead to further techniques that may be applied in tuberculosis drug discovery.The adoption of an 'either/or' approach to targeted or random tuberculosis drug screening is not expected. The historical success of random screening in providing the tuberculosis drugs currently in clinical use is likely to ensure that non-targeted protocols retain an important role in drug screening. However, a number of M. tuberculosis inhibitors in lead optimization and preclinical development have been discovered using targeted methods. Realization of the first clinically-approved tuberculosis drugs derived from targeted screening and continued refinements in targeted screening technologies are likely to increase the adoption of targeted approaches in the future.

A stereospecific synthesis of both enantiomers of the marine alkaloid eudistomin X using the amino acid chiral pool is achieved. Comparison of 1 H and 13 C NMR chemical shifts of the synthetic product as either the free base, mono-salt or disalt with those reported for the natural product established that the ascidian metabolite was originally characterised as a mono-salt and that the absolute configuration was (10R). 2010 Elsevier Ltd. All rights reserved.

BACKGROUND AND PURPOSE PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F420)-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA-824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis.

Background: The origin and spread of tuberculosis (TB) in Tasmania and the types of strains of Mycobacterium tuberculosis 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 Tasmania to better understand the epidemiology of TB in the state. Methods: Whole-genome sequencing was performed on cultured isolates of MTBC collected from 2014–2016. Single-locus variant analysis was applied to determine the phylogeny of the isolates and the presence of drug-resistance mutations. The genomic data were then cross-referenced against 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-Australian born individuals. Two possible TB clusters were identified based on single locus variant analysis, one from November-December 2014 (n = 2), with the second from MayAugust 2015 (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 mutations and potential overseas origin. In addition, we characterised a case of M. bovis TB in a Tasmanian-born person who presented in 2014, approximately 40 years after the last confirmed case in the state’s bovids. Conclusions: TB in Tasmania is predominantly of overseas origin with genotypically-unique drug-susceptible isolates of M. tuberculosis . However, the state also exhibits features of TB that are observed in other jurisdictions, namely, the clustering of cases, and drug resistance. Early detection of TB and contact tracing, particularly of overseas-born cases, coordinated with rapid laboratory drug-susceptibility testing and molecular typing, will be essential for Tasmania to reach the World Health Organisation’s TB eradication goals for low-incidence settings.

Many echinoderms contain subcuticular bacte- ria (SCB), symbionts which reside in the lumen between the host's epidermal cells and outer cuticle. This relationship is common, existing in about 60% of echinoderms studied so far, yet the function of SCB remains largely unknown. In this study, phylogenetic analysis was carried out on 16S rRNA sequences obtained from echinoderm-associated bac- teria, resulting in the identification of four species of puta- tive SCB. All four bacteria were identified from the ho- lothurian Stichopus mollis, and two of the four were also found in the asteroid Patiriella sp. Two of these bacteria belong to the Alphaproteobacteria, and two to the Gamma- proteobacteria. In addition to phylogenetic analysis, fluo- rescence in situ hybridization (FISH) assays were carried out on Patiriella sp., S. mollis, and the asteroid Astrostole scabra. Results showed that Patiriella sp. and S. mollis contain SCB, in agreement with the phylogenetic analysis, while SCB were not detected in A. scabra. Of the bacteria detected using FISH, more than 80% were recognized as belonging to the Alphaproteobacteria in both host species. However, in S. mollis about 20% of the detected SCB successfully hybridized with the Gammaproteobacteria- specific probe, whereas bacteria belonging to this class were never observed in Patiriella sp. This is only the second study to characterize SCB by molecular means, and is the first to identify SCB in situ using FISH.

In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection.