Whole genome sequencing of drug-resistant Mycobacterium tuberculosis isolates in Victoria, Australia.

OBJECTIVES: Whole genome sequencing (WGS) can identify clusters, transmission patterns, and drug resistance mutations. This is important in low-burden settings such as Australia, as it can assist in efficient contact tracing and surveillance. METHODS: We conducted a retrospective cohort study using WGS from 155 genomically defined drug-resistant Mycobacterium tuberculosis (DR-TB) isolates collected between 2018-2021 in Victoria, Australia. Bioinformatic analysis was performed to identify resistance-conferring mutations, lineages, clusters and understand how local sequences compared with international context. RESULTS: Of the 155 sequences, 42% were identified as lineage 2 and 35% as lineage 1; 65.8% (102/155) were isoniazid mono-resistant, 8.4% were multi-drug resistant TB and 5.8% were pre-extensively drug-resistant / extensively drug-resistant TB. The most common mutations were observed in katG and fabG1 genes, especially at Ser315Thr and fabG1 -15 C>T for first-line drugs. Ser450Leu was the most frequent mutation in rpoB gene. Phylogenetic analysis confirmed that Victorian DR-TB were associated with importation events. There was little evidence of local transmission with only five isolate pairs. CONCLUSION: Isoniazid-resistant TB is the commonest DR-TB in Victoria, and the mutation profile is similar to global circulating DR-TB. Most cases are diagnosed among migrants with limited transmission. This study highlights the value of WGS in identification of clusters and resistance-conferring mutations. This information is crucial in supporting disease mitigation and treatment strategies.

Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung.

The melioidosis bacterium, Burkholderia pseudomallei, is increasingly being recognised as a pathogen in patients with cystic fibrosis (CF). We have recently catalogued genome-wide variation of paired, isogenic B. pseudomallei isolates from seven Australasian CF cases, which were collected between 4 and 55 months apart. Here, we extend this investigation by documenting the transcriptomic changes in B. pseudomallei in five cases. Following growth in an artificial CF sputum medium, four of the five paired isolates exhibited significant differential gene expression (DE) that affected between 32 and 792 genes. The greatest number of DE events was observed between the strains from patient CF9, consistent with the hypermutator status of the latter strain, which is deficient in the DNA mismatch repair protein MutS. Two patient isolates harboured duplications that concomitantly increased expression of the ß-lactamase-encoding gene penA, and a 35 kb deletion in another abolished expression of 29 genes. Convergent expression profiles in the chronically-adapted isolates identified two significantly downregulated and 17 significantly upregulated loci, including the resistance-nodulation-division (RND) efflux pump BpeEF-OprC, the quorum-sensing hhqABCDE operon, and a cyanide- and pyocyanin-insensitive cytochrome bd quinol oxidase. These convergent pathoadaptations lead to increased expression of pathways that may suppress competing bacterial and fungal pathogens, and that enhance survival in oxygen-restricted environments, the latter of which may render conventional antibiotics less effective in vivo. Treating chronically adapted B. pseudomallei infections with antibiotics designed to target anaerobic infections, such as the nitroimidazole class of antibiotics, may significantly improve pathogen eradication attempts by exploiting this Achilles heel.

Raising the Stakes: Loss of Efflux Pump Regulation Decreases Meropenem Susceptibility in Burkholderia pseudomallei.

Background: Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug meropenem is the current gold standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance toward meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. Methods: We examined our 30-year clinical collection of melioidosis cases to identify B. pseudomallei isolates with reduced meropenem susceptibility. Isolates were subjected to minimum inhibitory concentration (MIC) testing toward meropenem. Paired isolates from patients who had evolved decreased susceptibility were subjected to whole-genome sequencing. Select agent-compliant genetic manipulation was carried out to confirm the molecular mechanisms conferring resistance. Results: We identified 11 melioidosis cases where B. pseudomallei isolates developed decreased susceptibility toward meropenem during treatment, including 2 cases not treated with this antibiotic. Meropenem MICs increased from 0.5-0.75 µg/mL to 3-8 µg/mL. Comparative genomics identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, with concomitant overexpression of their corresponding pumps. All cases were refractory to treatment despite aggressive, targeted therapy, and 2 were associated with a fatal outcome. Conclusions: This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. These findings have important ramifications for the diagnosis, treatment, and management of life-threatening melioidosis cases.

Individually tailored internet-based cognitive behaviour therapy for older adults with anxiety and depression: a randomised controlled trial.

Mixed anxiety and depression is common among older adults. The aim of the study was to compare the effects of an eight-week-long tailored internet-supported cognitive behaviour therapy (ICBT) programme and to compare against the provision of weekly general support. A second aim was to investigate if pre-treatment cognitive flexibility and self-reported cognitive problems would predict outcome. We included 66 older adults (aged over 60 years) with mixed anxiety/depression following media recruitment and randomised them into treatment and control groups. We also included a one-year follow-up. As a measure of executive function, we used the Wisconsin Card Sorting Test (perseverative errors) and the Cognitive Failures Questionnaire during the pre-treatment phase. Results showed a moderate between-group effect on the main outcome measure, the Beck Anxiety Inventory (BAI) (d= .50), favouring the treatment group. Nearly half (45.5%) of that group were classified as responders. One person (3%) in the treatment group deteriorated. There were significant correlations between perseverative errors and outcome (on the BAI r = -.45), but not among self-reported cognitive function. We conclude that guided, tailored ICBT may be effective for some older adults and that the role of cognitive function needs to be investigated further.

Within-Host Evolution of Burkholderia pseudomallei during Chronic Infection of Seven Australasian Cystic Fibrosis Patients.

Cystic fibrosis (CF) is a genetic disorder characterized by progressive lung function decline. CF patients are at an increased risk of respiratory infections, including those by the environmental bacterium Burkholderia pseudomallei, the causative agent of melioidosis. Here, we compared the genomes of B. pseudomallei isolates collected between ~4 and 55 months apart from seven chronically infected CF patients. Overall, the B. pseudomallei strains showed evolutionary patterns similar to those of other chronic infections, including emergence of antibiotic resistance, genome reduction, and deleterious mutations in genes involved in virulence, metabolism, environmental survival, and cell wall components. We documented the first reported B. pseudomallei hypermutators, which were likely caused by defective MutS. Further, our study identified both known and novel molecular mechanisms conferring resistance to three of the five clinically important antibiotics for melioidosis treatment. Our report highlights the exquisite adaptability of microorganisms to long-term persistence in their environment and the ongoing challenges of antibiotic treatment in eradicating pathogens in the CF lung. Convergent evolution with other CF pathogens hints at a degree of predictability in bacterial evolution in the CF lung and potential targeted eradication of chronic CF infections in the future.IMPORTANCEBurkholderia pseudomallei, the causative agent of melioidosis, is an environmental opportunistic bacterium that typically infects immunocompromised people and those with certain risk factors such as cystic fibrosis (CF). Patients with CF tend to develop chronic melioidosis infections, for reasons that are not well understood. This report is the first to describe B. pseudomallei evolution within the CF lung during chronic infection. We show that the pathways by which B. pseudomallei adapts to the CF lung are similar to those seen in better-studied CF pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia complex species. Adaptations include the accumulation of antibiotic resistance, loss of nonessential genes, metabolic alterations, and virulence factor attenuation. Known and novel mechanisms of resistance to three of the five antibiotics used in melioidosis treatment were identified. Similar pathways of evolution in CF pathogens, including B. pseudomallei, provide exciting avenues for more-targeted treatment of chronic, recalcitrant infections.

Whole-Genome Sequences of Five Burkholderia pseudomallei Isolates from Australian Cystic Fibrosis Patients.

We report here five improved high-quality draft genomes of Burkholderia pseudomallei isolated from Australian cystic fibrosis (CF) patients. This pathogen is rarely seen in CF patients. These genomes will be used to better understand chronic carriage of B. pseudomallei in the CF lung and the within-host evolution of longitudinal isolates from these patients.

Whole-genome sequencing of Burkholderia pseudomallei isolates from an unusual melioidosis case identifies a polyclonal infection with the same multilocus sequence type.

Twelve Burkholderia pseudomallei isolates collected over a 32-month period from a patient with chronic melioidosis demonstrated identical multilocus sequence types (STs). However, whole-genome sequencing suggests a polyclonal infection. This study is the first to report a mixed infection with the same ST.

An aspartic protease of the scabies mite Sarcoptes scabiei is involved in the digestion of host skin and blood macromolecules.

BACKGROUND: Scabies is a disease of worldwide significance, causing considerable morbidity in both humans and other animals. The scabies mite Sarcoptes scabiei burrows into the skin of its host, obtaining nutrition from host skin and blood. Aspartic proteases mediate a range of diverse and essential physiological functions such as tissue invasion and migration, digestion, moulting and reproduction in a number of parasitic organisms. We investigated whether aspartic proteases may play role in scabies mite digestive processes. METHODOLOGY/PRINCIPLE FINDINGS: We demonstrated the presence of aspartic protease activity in whole scabies mite extract. We then identified a scabies mite aspartic protease gene sequence and produced recombinant active enzyme. The recombinant scabies mite aspartic protease was capable of digesting human haemoglobin, serum albumin, fibrinogen and fibronectin, but not collagen III or laminin. This is consistent with the location of the scabies mites in the upper epidermis of human skin. CONCLUSIONS/SIGNIFICANCE: The development of novel therapeutics for scabies is of increasing importance given the evidence of emerging resistance to current treatments. We have shown that a scabies mite aspartic protease plays a role in the digestion of host skin and serum molecules, raising the possibility that interference with the function of the enzyme may impact on mite survival.

A diagnostic test for scabies: IgE specificity for a recombinant allergen of Sarcoptes scabiei.

Scabies infestations are difficult to diagnose clinically and current serologic tests have less than 50% accuracy. To develop more reliable diagnosis of scabies, specific IgE antibodies to a major scabies antigen recombinant Sar s 14.3 (rSar s 14.3) were measured in 140 plasma samples from scabies-infested and control subject groups using dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA). Levels of rSar s 14.3-specific IgE were quantified, and cross-reactivity with its house dust mite homologue, Der p 14, was assessed. The rSar s 14.3 DELFIA showed excellent diagnostic capability, with 100% sensitivity and 93.75% specificity for distinguishing subjects with current scabies infestation from control, uninfested subjects. Recombinant Der p 14 preparation was ineffective at inhibiting IgE binding to rSar s 14.3. This study shows that quantification of levels of IgE antibody to rSar s 14.3 is a highly sensitive method for diagnosis of scabies infestation in clinical practice.

Increased allergic immune response to Sarcoptes scabiei antigens in crusted versus ordinary scabies.

Scabies, a parasitic skin infestation by the burrowing "itch" mite Sarcoptes scabiei, causes significant health problems for children and adults worldwide. Crusted scabies is a particularly severe form of scabies in which mites multiply into the millions, causing extensive skin crusting. The symptoms and signs of scabies suggest host immunity to the scabies mite, but the specific resistant response in humans remains largely uncharacterized. We used 4 scabies mite recombinant proteins with sequence homology to extensively studied house dust mite allergens to investigate a differential immune response between ordinary scabies and the debilitating crusted form of the disease. Subjects with either disease form showed serum IgE against recombinant S. scabiei cysteine and serine proteases and apolipoprotein, whereas naive subjects showed minimal IgE reactivity. Significantly (P < 0.05) greater serum IgE and IgG4 binding to mite apolipoprotein occurred in subjects with crusted scabies than in those with ordinary scabies. Both subject groups showed strong proliferative responses (peripheral blood mononuclear cells) to the scabies antigens, but the crusted scabies group showed increased secretion of the Th2 cytokines interleukin 5 (IL-5) and IL-13 and decreased Th1 cytokine gamma interferon (IFN-gamma) in response to the active cysteine protease. These data confirm that a nonprotective allergic response occurs in the crusted disease form and demonstrate that clinical severity is associated with differences in the type and magnitude of the antibody and cellular responses to scabies proteins. A quantitative IgE inhibition assay identified IgE immunoreactivity of scabies mite antigens distinct from that of house dust mite antigens, which is potentially important for specific scabies diagnosis and therapy.