Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine ...hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se.
•Genomic signatures of host-adaptation and virulence of Salmonella are emerging.•Sequence analyses can aid the prediction of zoonotic risk and source attribution.•Host-specific, niche-specific and ...variant-specific virulence factors have been identified.•Comparative and functional genomics identify options for control of Salmonella.•Rodent and cell-based assays do not always reflect salmonellosis in farm animals.
Salmonella is an animal and zoonotic pathogen of global importance. Depending on pathogen and host factors, infections can be asymptomatic or involve acute gastroenteritis or invasive disease. Genomic signatures associated with host-range, tissue tropism or differential virulence of Salmonella enterica serovars, and their variants, have emerged. In turn, it is becoming feasible to predict invasive potential, host-adaptation and zoonotic risk of Salmonella from sequence data to improve outbreak investigation, risk assessment and control strategies. Functional annotation of Salmonella genomes has accelerated with the screening of high-density mutant libraries, revealing host-specific, niche-specific and serovar-specific virulence factors. As natural hosts and reservoirs, farmed animals provide powerful insights into host-adaptation and pathogenesis of Salmonella not always evident from surrogate rodent or cell-based models.
Avian pathogenic Escherichia coli (APEC) cause colibacillosis in birds, a syndrome of severe respiratory and systemic disease that constitutes a major threat due to early mortality, condemnation of ...carcasses and reduced productivity. APEC can infect different types of birds in all commercial settings, and birds of all ages, although disease tends to be more severe in younger birds likely a consequence of an immature immune system. APEC can act as both primary and secondary pathogens, with predisposing factors for secondary infections including poor housing conditions, respiratory viral and Mycoplasma spp. infections or vaccinations. Controlled studies with APEC as primary pathogens have been used to study the bird's immune response to APEC, although it may not always be representative of natural infections which may be more complex due to the presence of secondary agents, stress and environmental factors. Under controlled experimental conditions, a strong early innate immune response is induced which includes host defence peptides in mucus and a cellular response driven by heterophils and macrophages. Both antibody and T-cell mediated adaptive responses have been demonstrated after vaccination. In this review we will discuss the bird's immune response to APEC as primary pathogen with a bias towards the innate immune response, as mechanistic adaptive studies clearly form a much more limited body of work despite numerous vaccine trials.
Microglia have critical roles in neural development, homeostasis and neuroinflammation and are increasingly implicated in age-related neurological dysfunction. Neurodegeneration often occurs in ...disease-specific, spatially restricted patterns, the origins of which are unknown. We performed to our knowledge the first genome-wide analysis of microglia from discrete brain regions across the adult lifespan of the mouse, and found that microglia have distinct region-dependent transcriptional identities and age in a regionally variable manner. In the young adult brain, differences in bioenergetic and immunoregulatory pathways were the major sources of heterogeneity and suggested that cerebellar and hippocampal microglia exist in a more immune-vigilant state. Immune function correlated with regional transcriptional patterns. Augmentation of the distinct cerebellar immunophenotype and a contrasting loss in distinction of the hippocampal phenotype among forebrain regions were key features during aging. Microglial diversity may enable regionally localized homeostatic functions but could also underlie region-specific sensitivities to microglial dysregulation and involvement in age-related neurodegeneration.
The cecal microbiota plays numerous roles in chicken health and nutrition. Where such microbiota differs between lines exhibiting distinct phenotypes, microbiota transplantation offers scope to ...dissect the role of gut microbial communities in those traits. However, the composition and stability of transplants over time is relatively ill-defined and varying levels of success have been reported. In this study, we transplanted cecal contents from adult Roslin broilers into chicks from a different broiler line. Within <12 h posthatch, Ross 308 chicks received an oral gavage of cecal contents (n = 26) or a PBS control (n = 24). Cecal contents samples were collected postmortem from birds on d 1, 2, 3, 4, and 7 posthatch. DNA was extracted from these samples and the transplant inoculum and the V4 region of the 16S rRNA gene was amplified and sequenced. The cecal microbiota of chickens receiving the microbiota transplant was significantly different in composition and significantly richer and more diverse, in comparison to control birds. At the final timepoint (d 7), of the 150 Operational Taxonomic Units (OTUs) that were >0.1% abundant (average) in the donor sample, 137 were detected in the treated group (75 were >0.1% abundant (average)) while only 88 were detected in the control group (29 were >0.1% abundant (average)). Our data therefore suggests that stable transplantation of the cecal microbiota between lines is achievable using the methods described in this paper.
Two lineages of Salmonella enterica serovar Typhimurium (S. Typhimurium) of multi-locus sequence type ST313 have been linked with the emergence of invasive Salmonella disease across sub-Saharan ...Africa. The expansion of these lineages has a temporal association with the HIV pandemic and antibiotic usage. We analysed the whole genome sequence of 129 ST313 isolates representative of the two lineages and found evidence of lineage-specific genome degradation, with some similarities to that observed in S. Typhi. Individual ST313 S. Typhimurium isolates exhibit a distinct metabolic signature and modified enteropathogenesis in both a murine and cattle model of colitis, compared to S. Typhimurium outside of the ST313 lineages. These data define phenotypes that distinguish ST313 isolates from other S. Typhimurium and may represent adaptation to a distinct pathogenesis and lifestyle linked to an-immuno-compromised human population.
Avian colibacillosis is caused by a group of pathogens designated avian pathogenic Escherichia coli (APEC). Despite being known for over a century, avian colibacillosis remains one of the major ...endemic diseases afflicting the poultry industry worldwide. Autologous bacterins provide limited serotype-specific protection, yet multiple serogroups are associated with disease, especially O1, O2 and O78 among many others. Experimental infection models have facilitated the identification of some key APEC virulence genes and have allowed testing of vaccine candidates. Well-recognized virulence factors include Type 1 (F1) and P (Pap/Prs) fimbriae for colonization, IbeA for invasion, iron acquisition systems, TraT and Iss for serum survival, K and O antigens for anti-phagocytic activity, and a temperature-sensitive haemagglutinin of imprecise function. Intriguingly, these factors do not occur universally among APEC, suggesting the presence of multiple alternative mechanisms mediating pathogenicity. The recent availability of the first complete APEC genome sequence can be expected to accelerate the identification of bacterial genes expressed during infection and required for virulence. High-throughput molecular approaches like signature-tagged transposon mutagenesis have already proved invaluable in revealing portfolios of genes expressed by pathogenic bacteria during infection, and this has enabled identification of APEC O2 factors required for septicaemia in the chicken model. Complimentary approaches, such as in vivo-induced antigen technology, exist to define the activities of APEC in vivo. In recent years, reverse vaccinology and immuno-proteomic approaches have also enabled identification of novel vaccine candidates in other bacterial pathogens. Collectively, such information provides the basis for the development or improvement of strategies to control APEC infections in the food-producing avian species.
Summary Background In South Africa, sputum smear microscopy has been replaced with Xpert MTB/RIF as the initial diagnostic test for tuberculosis. In a pragmatic parallel cluster-randomised trial, we ...evaluated the effect on patient and programme outcomes. Methods We randomly allocated 20 laboratories (clusters) in medium-burden districts of South Africa to either an Xpert (immediate Xpert) or microscopy (Xpert deferred) group (1:1), stratified by province. At two primary care clinics per laboratory, a systematic sample of adults giving sputum for tuberculosis investigation was assessed for eligibility. The primary outcome was mortality at 6 months from enrolment. Masking of participants' group allocation was not possible because of the pragmatic trial design. The trial is registered with the ISRCTN registry (ISRCTN68905568) and the South African Clinical Trial Register (DOH-27-1011-3849). Findings Between June and November, 2012, 4972 people were screened, and 4656 (93·6%) enrolled (median age 36 years; 2891 62% female; 2212 62% reported being HIV-positive). There was no difference between the Xpert and microscopy groups with respect to mortality at 6 months (91/2324 3·9% vs 116/2332 5·0%, respectively; adjusted risk ratio aRR 1·10, 95% CI 0·75–1·62). Interpretation Xpert did not reduce mortality at 6 months compared with sputum microscopy. Improving outcomes in drug-sensitive tuberculosis programmes might require not only better diagnostic tests but also better linkage to care. Funding Bill & Melinda Gates Foundation.
To address the need for sensitive high-throughput assays to analyse avian innate and adaptive immune responses, we developed and validated a highly multiplexed qPCR 96.96 Fluidigm Dynamic Array to ...analyse the transcription of chicken immune-related genes. This microfluidic system permits the simultaneous analysis of expression of 96 transcripts in 96 samples in 6 nanolitre reactions and the 9,216 reactions are ready for interpretation immediately. A panel of 89 genes was selected from an RNA-seq analysis of the transcriptional response of chicken macrophages, dendritic cells and heterophils to agonists of innate immunity and from published transcriptome data. Assays were confirmed to be highly specific by amplicon sequencing and melting curve analysis and the reverse transcription and preamplification steps were optimised. The array was applied to RNA of various tissues from a commercial line of broiler chickens housed at two different levels of biosecurity. Gut-associated lymphoid tissues, bursa, spleen and peripheral blood leukocytes were isolated and transcript levels for immune-related genes were defined. The results identified blood cells as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially transcribed between birds housed under varying biosecurity levels. Conventional qPCR analysis of three differentially transcribed genes confirmed the results from the multiplex qPCR array. A highly multiplexed qPCR-based platform for evaluation of chicken immune responses has been optimised and validated using samples from commercial chickens. Apart from applications in selective breeding programmes, the array could be used to analyse the complex interplay between the avian immune system and pathogens by including pathogen-specific probes, to screen vaccine responses, and as a predictive tool for immune robustness.
The Xpert MTB/RIF assay is an automated molecular test that has improved the detection of tuberculosis and rifampicin resistance, but its sensitivity is inadequate in patients with paucibacillary ...disease or HIV. Xpert MTB/RIF Ultra (Xpert Ultra) was developed to overcome this limitation. We compared the diagnostic performance of Xpert Ultra with that of Xpert for detection of tuberculosis and rifampicin resistance.
In this prospective, multicentre, diagnostic accuracy study, we recruited adults with pulmonary tuberculosis symptoms presenting at primary health-care centres and hospitals in eight countries (South Africa, Uganda, Kenya, India, China, Georgia, Belarus, and Brazil). Participants were allocated to the case detection group if no drugs had been taken for tuberculosis in the past 6 months or to the multidrug-resistance risk group if drugs for tuberculosis had been taken in the past 6 months, but drug resistance was suspected. Demographic information, medical history, chest imaging results, and HIV test results were recorded at enrolment, and each participant gave at least three sputum specimen on 2 separate days. Xpert and Xpert Ultra diagnostic performance in the same sputum specimen was compared with culture tests and drug susceptibility testing as reference standards. The primary objectives were to estimate and compare the sensitivity of Xpert Ultra test with that of Xpert for detection of smear-negative tuberculosis and rifampicin resistance and to estimate and compare Xpert Ultra and Xpert specificities for detection of rifampicin resistance. Study participants in the case detection group were included in all analyses, whereas participants in the multidrug-resistance risk group were only included in analyses of rifampicin-resistance detection.
Between Feb 18, and Dec 24, 2016, we enrolled 2368 participants for sputum sampling. 248 participants were excluded from the analysis, and 1753 participants were distributed to the case detection group (n=1439) and the multidrug-resistance risk group (n=314). Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0). Specificities of Xpert Ultra and Xpert for case detection were 96% and 98% (−2·7%, −3·9 to −1·7) overall, and 93% and 98% for patients with a history of tuberculosis. Xpert Ultra and Xpert performed similarly in detecting rifampicin resistance.
For tuberculosis case detection, sensitivity of Xpert Ultra was superior to that of Xpert in patients with paucibacillary disease and in patients with HIV. However, this increase in sensitivity came at the expense of a decrease in specificity.
Government of Netherlands, Government of Australia, Bill & Melinda Gates Foundation, Government of the UK, and the National Institute of Allergy and Infectious Diseases.
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