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Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T cell concentration is associated with higher mortality even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. Additionally, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during a severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.
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Melioidosis is an often fatal infection in tropical regions caused by an environmental bacterium, Burkholderia pseudomallei Current recommended melioidosis treatment requires intravenous ß-lactam antibiotics such as ceftazidime (CAZ), meropenem (MEM) or amoxicillin-clavulanic acid (AMC) and oral trimethoprim-sulfamethoxazole. Emerging antibiotic resistance could lead to therapy failure and high mortality. We performed a prospective multicentre study in northeast Thailand during 2015-2018 to evaluate antibiotic susceptibility and characterize ß-lactam resistance in clinical B. pseudomallei isolates. Collection of 1,317 B. pseudomallei isolates from patients with primary and relapse infections were evaluated for susceptibility to CAZ, imipenem (IPM), MEM and AMC. ß-lactam resistant isolates were confirmed by broth microdilution method and characterized by whole genome sequence analysis, penA expression and ß-lactamase activity. The resistant phenotype was verified via penA mutagenesis. All primary isolates were IPM-susceptible but we observed two CAZ-resistant and one CAZ-intermediate resistant isolates, two MEM-less susceptible isolates, one AMC-resistant and two AMC-intermediate resistant isolates. One of 13 relapse isolates was resistant to both CAZ and AMC. Two isolates were MEM-less susceptible. Strains DR10212A (primary) and DR50054E (relapse) were multi-drug resistant. Genomic and mutagenesis analyses supplemented with gene expression and ß-lactamase analyses demonstrated that CAZ-resistant phenotype was caused by PenA variants: P167S (N=2) and penA amplification (N=1). Despite the high mortality rate in melioidosis, our study revealed that B. pseudomallei isolates had a low frequency of ß-lactam resistance caused by penA alterations. Clinical data suggest that resistant variants may emerge in patients during antibiotic therapy and be associated with poor response to treatment.
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Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.
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Burkholderia pseudomallei , Melioidosis , Humanos , Melioidosis/diagnóstico , Melioidosis/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Anticuerpos Antibacterianos , Burkholderia pseudomallei/genética , Sensibilidad y Especificidad , Proteínas Hemolisinas/genética , Pruebas Diagnósticas de RutinaRESUMEN
BACKGROUND: Melioidosis, infection caused by Burkholderia pseudomallei, is a common cause of sepsis with high associated mortality in Southeast Asia. Identification of patients at high likelihood of clinical deterioration is important for guiding decisions about resource allocation and management. We sought to develop a biomarker-based model for 28-day mortality prediction in melioidosis. METHODS: In a derivation set (Nâ =â 113) of prospectively enrolled, hospitalized Thai patients with melioidosis, we measured concentrations of interferon-γ, interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-É, granulocyte-colony stimulating factor, and interleukin-17A. We used least absolute shrinkage and selection operator (LASSO) regression to identify a subset of predictive biomarkers and performed logistic regression and receiver operating characteristic curve analysis to evaluate biomarker-based prediction of 28-day mortality compared with clinical variables. We repeated select analyses in an internal validation set (Nâ =â 78) and in a prospectively enrolled external validation set (Nâ =â 161) of hospitalized adults with melioidosis. RESULTS: All 8 cytokines were positively associated with 28-day mortality. Of these, interleukin-6 and interleukin-8 were selected by LASSO regression. A model consisting of interleukin-6, interleukin-8, and clinical variables significantly improved 28-day mortality prediction over a model of only clinical variables [AUC (95% confidence interval [CI]): 0.86 (.79-.92) vs 0.78 (.69-.87); Pâ =â .01]. In both the internal validation set (0.91 [0.84-0.97]) and the external validation set (0.81 [0.74-0.88]), the combined model including biomarkers significantly improved 28-day mortality prediction over a model limited to clinical variables. CONCLUSIONS: A 2-biomarker model augments clinical prediction of 28-day mortality in melioidosis.
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Citocinas/sangre , Melioidosis , Adulto , Biomarcadores/sangre , Burkholderia pseudomallei , Humanos , Melioidosis/diagnóstico , Melioidosis/mortalidad , TailandiaRESUMEN
Melioidosis is a tropical infection caused by the intracellular pathogen Burkholderia pseudomallei, an underreported and emerging global threat. As melioidosis-associated mortality is frequently high despite antibiotics, novel management strategies are critically needed. Therefore, we sought to determine whether functional changes in the host innate and adaptive immune responses are induced during acute melioidosis and are associated with outcome. Using a unique whole blood stimulation assay developed for use in resource-limited settings, we examined induced cellular functional and phenotypic changes in a cohort of patients with bacteremic melioidosis prospectively enrolled within 24 h of positive blood culture and followed for 28 days. Compared to healthy controls, melioidosis survivors generated an IL-17 response mediated by Th17 cells and terminally-differentiated effector memory CD8+ T cells (P < .05, both), persisting to 28 days after enrolment. Furthermore, melioidosis survivors developed polyfunctional cytokine production in CD8+ T cells (P < .01). Conversely, a reduction in CCR6+ CD4+ T cells was associated with higher mortality, even after adjustments for severity of illness (P = 0.004). Acute melioidosis was also associated with a profound acute impairment in monocyte function as stimulated cytokine responses were reduced in classical, intermediate and non-classical monocytes. Impaired monocyte cytokine function improved by 28-days after enrolment. These data suggest that IL-17 mediated cellular responses may be contributors to host defense during acute melioidosis, and that innate immune function may be impaired. These insights could provide novel targets for the development of therapies and vaccine targets in this frequently lethal disease.
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Burkholderia pseudomallei , Linfocitos T CD8-positivos , Melioidosis , Células Th17 , Melioidosis/inmunología , Melioidosis/mortalidad , Melioidosis/microbiología , Humanos , Masculino , Femenino , Burkholderia pseudomallei/inmunología , Persona de Mediana Edad , Linfocitos T CD8-positivos/inmunología , Células Th17/inmunología , Anciano , Adulto , Inmunidad Celular , Interleucina-17/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/sangre , Citocinas/inmunología , Estudios ProspectivosRESUMEN
Objective: Unique metabolic requirements accompany the development and functional fates of immune cells. How cellular metabolism is important in natural killer (NK) cells and their memory-like differentiation in bacterial infections remains elusive. Methods: Here, we utilise our established NK cell memory assay to investigate the metabolic requirement for memory-like NK cell formation and function in response to the Gram-negative intracellular bacteria Burkholderia pseudomallei (BP), the causative agent of melioidosis. Results: We demonstrate that CD160+ memory-like NK cells upon BP stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients which is maintained at least 3-month post-hospital admission. Using an in vitro assay, human BP-specific CD160+ memory-like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake, increased mTOR activation and mitochondrial membrane potential upon BP re-stimulation. Antigen-specific and cytokine-induced IFN-γ production of this memory-like NK cell subset are highly dependent on oxidative phosphorylation (OXPHOS) with some dependency on glycolysis, whereas the formation of CD160+ memory-like NK cells in vitro is dependent on fatty acid oxidation and OXPHOS and further increased by metformin. Conclusion: This study reveals the link between metabolism and cellular function of memory-like NK cells, which can be exploited for vaccine design and for monitoring protection against Gram-negative bacterial infection.
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Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conduct a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identifies three dominant lineages, each with unique gene sets potentially enhancing bacterial fitness in the environment. We find that recombination drives lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage reveal increased expression of lineage-specific genes under environmental conditions in two out of three lineages. This underscores the potential importance of environmental persistence for these dominant lineages. The study also highlights the influence of environmental factors such as terrain slope, altitude, and river direction on the geographical dispersal of B. pseudomallei. Collectively, our findings suggest that environmental persistence may play a role in facilitating the spread of B. pseudomallei, and as a prerequisite for exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.
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Burkholderia pseudomallei , Variación Genética , Melioidosis , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/aislamiento & purificación , Burkholderia pseudomallei/clasificación , Melioidosis/microbiología , Melioidosis/epidemiología , Tailandia/epidemiología , Humanos , Filogenia , Flujo Génico , Genoma Bacteriano/genéticaRESUMEN
Melioidosis, infection caused by Burkholderia pseudomallei, is characterized by robust innate immune responses. We have previously reported associations of TLR1 single nucleotide missense variant rs76600635 with mortality and of TLR5 nonsense variant rs5744168 with both bacteremia and mortality in single-center studies of patients with melioidosis in northeastern Thailand. The objective of this study was to externally validate the associations of rs76600635 and rs5744168 with bacteremia and mortality in a large multicenter cohort of melioidosis patients. We genotyped rs76600635 and rs5744168 in 1,338 melioidosis patients enrolled in a prospective parent cohort study conducted at nine hospitals in northeastern Thailand. The genotype frequencies of rs76600635 did not differ by bacteremia status (P = 0.27) or 28-day mortality (P = 0.84). The genotype frequencies of rs5744168 did not differ by either bacteremia status (P = 0.46) or 28-day mortality (P = 0.10). Assuming a dominant genetic model, there was no association of the rs76600635 variant with bacteremia (adjusted odds ratio [OR], 0.75; 95% CI, 0.54-1.04, P = 0.08) or 28-day mortality (adjusted OR, 0.96; 95% CI, 0.71-1.28, P = 0.77). There was no association of the rs5744168 variant with bacteremia (adjusted OR, 1.24; 95% CI, 0.76-2.03, P = 0.39) or 28-day mortality (adjusted OR, 1.22; 95% CI, 0.83-1.79, P = 0.21). There was also no association of either variant with 1-year mortality. We conclude that in a large multicenter cohort of patients hospitalized with melioidosis in northeastern Thailand, neither TLR1 missense variant rs76600635 nor TLR5 nonsense variant rs5744168 is associated with bacteremia or mortality.
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Bacteriemia , Melioidosis , Receptor Toll-Like 1 , Receptor Toll-Like 5 , Humanos , Melioidosis/mortalidad , Melioidosis/genética , Melioidosis/microbiología , Masculino , Femenino , Receptor Toll-Like 1/genética , Tailandia/epidemiología , Persona de Mediana Edad , Bacteriemia/mortalidad , Bacteriemia/microbiología , Bacteriemia/genética , Receptor Toll-Like 5/genética , Adulto , Estudios de Cohortes , Polimorfismo de Nucleótido Simple , Genotipo , Burkholderia pseudomallei/genética , Estudios Prospectivos , Anciano , Predisposición Genética a la EnfermedadRESUMEN
Rationale: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin) use is associated with a lower risk of incident pneumonia and, less robustly, with nonpulmonary infections. Whether statin use is associated with a lower risk of pneumonia than other clinical presentations of infection with the same pathogen is unknown. Objectives: To assess whether preadmission statin use is associated with a lower risk of pneumonia than nonpneumonia presentations among patients hospitalized with Burkholderia pseudomallei infection (melioidosis). Methods: We performed a secondary analysis of a prospective multicenter cohort study of patients hospitalized with culture-confirmed B. pseudomallei infection (melioidosis). We used Poisson regression with robust standard errors to test for an association between statin use and pneumonia. We then performed several sensitivity analyses that addressed healthy user effect and indication bias. Results: Of 1,372 patients with melioidosis enrolled in the parent cohort, 1,121 were analyzed. Nine hundred eighty (87%) of 1,121 were statin nonusers, and 141 (13%) of 1,121 were statin users. Forty-six (33%) of 141 statin users presented with pneumonia compared with 432 (44%) of 980 statin nonusers. Statin use was associated with a lower risk of pneumonia in unadjusted analysis (relative risk, 0.74; 95% confidence interval, 0.58-0.95; P = 0.02) and, after adjustment for demographic variables, comorbidities, environmental exposures, and symptom duration (relative risk, 0.73; 95% confidence interval, 0.57-0.94; P = 0.02). The results of sensitivity analyses, including active comparator analysis and inverse probability of treatment weighting, were consistent with the primary analysis. Conclusions: In hospitalized patients with melioidosis, preadmission statin use was associated with a lower risk of pneumonia than other clinical presentations of melioidosis, suggesting a lung-specific protective effect of statins.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Melioidosis , Neumonía , Humanos , Melioidosis/tratamiento farmacológico , Melioidosis/epidemiología , Melioidosis/inducido químicamente , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Estudios de Cohortes , Estudios Prospectivos , Neumonía/complicaciones , PulmónRESUMEN
Melioidosis, a neglected tropical infection caused by Burkholderia pseudomallei, commonly presents as pneumonia or sepsis with mortality rates up to 50% despite appropriate treatment. A better understanding of the early host immune response to melioidosis may lead to new therapeutic interventions and prognostication strategies to reduce disease burden. Whole blood transcriptomic signatures in 164 melioidosis patients and 70 patients with other infections hospitalized in northeastern Thailand enrolled within 24 hours following hospital admission were studied. Key findings were validated in an independent melioidosis cohort. Melioidosis was characterized by upregulation of interferon signaling responses compared to other infections. Mortality in melioidosis was associated with excessive inflammation, up-regulated type 2 immune responses and a dramatic decrease in T cell-mediated immunity compared to survivors. We identified and independently confirmed a five-gene predictive set classifying fatal melioidosis (validation cohort: an area under the receiver operating characteristic curve 0.83, 95% CI: 0.67-0.99). In conclusion, this study highlights the intricate balance between innate and adaptive immunity during fatal melioidosis and can inform future precision medicine strategies for targeted therapies and prognostication in this severe infection.
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The humoral immune response plays a key role in protecting the population from SARS-CoV-2 transmission. Patients who recovered from COVID-19 as well as fully vaccinated individuals have elevated levels of antibodies. The dynamic levels of the classes and subclasses of antibody responses to new variants that occur in different populations remain unclear. We prospectively recruited 60 participants, including COVID-19 patients and CoronaVac-vaccinated individuals, in Thailand from May to August 2021. Plasma samples were collected on day 0, day 14, and day 28 to determine the dynamic levels of the classes and subclasses of plasma antibodies against the receptor-binding domain (RBD) in the spike protein (S) of four SARS-CoV-2 strains (Wuhan, Alpha, Delta, and Omicron) via enzyme-linked immunosorbent assay. Our results indicated that the patients with SARS-CoV-2 infections had broader class and subclass profiles as well as higher levels of anti-S RBD antibodies to the Wuhan, Alpha, and Delta strains than did the CoronaVac-vaccinated individuals. The median antibody levels increased and subsequently declined in a month in the COVID-19 patients and in the vaccinated group. Correlations of the classes and subclasses of antibodies were observed in the COVID-19 patients but not in the vaccinated individuals. The levels of all of the anti-S RBD antibodies against the Omicron variant were low in the patients and in the vaccinated individuals. Our study revealed distinct antibody profiles between the two cohorts, suggesting different pathways of immune activation. This could have an impact on protection from infections by new variants of concern (VOC). IMPORTANCE The antibody responses to new SARS-CoV-2 variants that occur in different populations remain unclear. In this study, we recruited 60 participants, including COVID-19 patients and CoronaVac-vaccinated individuals, in Thailand and determined the dynamic levels of the IgG, IgA, IgM, and IgG subclasses of antibodies against the spike protein (S) of four SARS-CoV-2 strains. Our results showed that the patients with SARS-CoV-2 infections had broader profiles and higher levels of antibodies to the Wuhan, Alpha, and Delta strains than did the CoronaVac-vaccinated individuals. The antibody levels of both groups increased and subsequently decreased within 1 month. Higher and functional correlations of these antibodies were observed in the COVID-19 patients. The levels of all anti-S RBD antibodies against the Omicron variant were low in patients and vaccinated individuals. Our study revealed distinct antibody responses between the two groups, suggesting different pathways of immune response, which may have an impact on protection from infections by new SARS-CoV-2 variants.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevención & control , Formación de Anticuerpos , Tailandia , Glicoproteína de la Espiga del Coronavirus , Inmunoglobulina G , VacunaciónRESUMEN
The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.
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Introduction: Melioidosis is an often-fatal tropical infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, but few studies have identified promising biomarker candidates to predict outcome. Methods: In 78 prospectively enrolled patients hospitalized with melioidosis, six candidate protein biomarkers, identified from the literature, were measured in plasma at enrollment. A multi-biomarker model was developed using least absolute shrinkage and selection operator (LASSO) regression, and mortality discrimination was compared to a clinical variable model by receiver operating characteristic curve analysis. Mortality prediction was confirmed in an external validation set of 191 prospectively enrolled patients hospitalized with melioidosis. Results: LASSO regression selected IL-1R2 and soluble triggering receptor on myeloid cells 1 (sTREM-1) for inclusion in the candidate biomarker model. The areas under the receiver operating characteristic curve (AUC) for mortality discrimination for the IL-1R2 + sTREM-1 model (AUC 0.81, 95% CI 0.72-0.91) as well as for an IL-1R2-only model (AUC 0.78, 95% CI 0.68-0.88) were higher than for a model based on a modified Sequential Organ Failure Assessment (SOFA) score (AUC 0.69, 95% CI 0.56-0.81, p < 0.01, p = 0.03, respectively). In the external validation set, the IL-1R2 + sTREM-1 model (AUC 0.86, 95% CI 0.81-0.92) had superior 28-day mortality discrimination compared to a modified SOFA model (AUC 0.80, 95% CI 0.74-0.86, p < 0.01) and was similar to a model containing IL-1R2 alone (AUC 0.82, 95% CI 0.76-0.88, p = 0.33). Conclusion: Biomarker models containing IL-1R2 had improved 28-day mortality prediction compared to clinical variable models in melioidosis and may be targets for future, rapid test development.
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NK cells are endowed with immunological memory to a range of pathogens but the development of NK cell memory in bacterial infections remains elusive. Here, we establish an assay inducing memory-like NK cell response to Burkholderia pseudomallei, the causative agent of the severe bacterial disease called melioidosis, and explore NK cell memory in a melioidosis patient cohort. We show that NK cells require bacteria-primed monocytes to acquire memory-like properties, demonstrated by bacteria-specific responses, features that strongly associate with CD160 expression. Induction of this memory-like NK cell is partly dependent on CD160 and IL-12R. Importantly, CD160 expression identifies memory-like NK cells in a cohort of recovered melioidosis patients with heightened responses maintained at least 3 months post hospital admission and reduced numbers of this cell population independently correlate with recurrent melioidosis. These newly identified memory-like NK cells are a promising target for future vaccine design and for monitoring protection against infection.
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Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conducted a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identified three dominant lineages with unique gene sets enhancing bacterial fitness, indicating lineage-specific adaptation strategies. Crucially, recombination was found to drive lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage revealed heightened expression of lineage-specific genes in environmental versus infection conditions, notably under nutrient depletion, highlighting environmental persistence as a key factor in the success of dominant lineages. The study also revealed the role of environmental factors - slope of terrain, altitude, direction of rivers, and the northeast monsoons - in shaping B. pseudomallei geographical dispersal. Collectively, our findings highlight persistence in the environment as a pivotal element facilitating B. pseudomallei spread, and as a prelude to exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.
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Background: Melioidosis is a neglected tropical infection caused by the environmental saprophyte Burkholderia pseudomallei. Methods: We conducted a prospective, observational study at nine hospitals in northeastern Thailand, a hyperendemic melioidosis zone, to define current characteristics of melioidosis patients and quantify outcomes over one year. Findings: 2574 individuals hospitalised with culture-confirmed melioidosis were screened and 1352 patients were analysed. The median age was 55 years, 975 (72%) were male, and 951 (70%) had diabetes. 565 (42%) patients presented with lung infection, 1042 (77%) were bacteremic, 442 (33%) received vasopressors/inotropes and 547 (40%) received mechanical ventilation. 1307 (97%) received an intravenous antibiotic against B. pseudomallei. 335/1345 (25%) patients died within one month and 448/1322 (34%) of patients died within one year. Most patients had risk factors for melioidosis, but patients without identified risk factors did not have a reduced risk of death. Of patients discharged alive, most received oral trimethoprim-sulfamethoxazole, which was associated with decreased risk of post-discharge death; 235/970 (24%) were readmitted, and 874/1015 (86%) survived to one year. Recurrent infection was detected in 17/994 patients (2%). Patients with risk factors other than diabetes had increased risk of death and increased risk of hospital readmission. Interpretation: In northeastern Thailand patients with melioidosis experience high rates of bacteremia, organ failure and death. Most patients discharged alive survive one year although all-cause readmission is common. Recurrent disease is rare. Strategies that emphasize prevention, rapid diagnosis and intensification of early clinical management are likely to have greatest impact in this and other resource-restricted regions. Funding: US NIH/NIAID U01AI115520.
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Melioidosis is an often lethal tropical disease caused by the Gram-negative bacillus, Burkholderia pseudomallei. The study objective was to characterize transcriptomes in melioidosis patients and identify genes associated with outcome. Whole blood RNA-seq was performed in a discovery set of 29 melioidosis patients and 3 healthy controls. Transcriptomic profiles of patients who did not survive to 28 days were compared with patients who survived and healthy controls, showing 65 genes were significantly up-regulated and 218 were down-regulated in non-survivors compared to survivors. Up-regulated genes were involved in myeloid leukocyte activation, Toll-like receptor cascades and reactive oxygen species metabolic processes. Down-regulated genes were hematopoietic cell lineage, adaptive immune system and lymphocyte activation pathways. RT-qPCR was performed for 28 genes in a validation set of 60 melioidosis patients and 20 healthy controls, confirming differential expression. IL1R2, GAS7, S100A9, IRAK3, and NFKBIA were significantly higher in non-survivors compared with survivors (P < 0.005) and healthy controls (P < 0.0001). The AUROCC of these genes for mortality discrimination ranged from 0.80-0.88. In survivors, expression of IL1R2, S100A9 and IRAK3 genes decreased significantly over 28 days (P < 0.05). These findings augment our understanding of this severe infection, showing expression levels of specific genes are potential biomarkers to predict melioidosis outcomes.
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Biomarcadores/sangre , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Melioidosis/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Melioidosis/sangre , Melioidosis/genética , Persona de Mediana Edad , Estudios Prospectivos , Análisis de Secuencia de ARN , Análisis de SupervivenciaRESUMEN
BACKGROUND: Burkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood. METHODOLOGY/PRINCIPLE FINDINGS: To gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes. CONCLUSIONS/SIGNIFICANCE: Our plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection.
Asunto(s)
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/aislamiento & purificación , Genoma Bacteriano/genética , Células Gigantes/microbiología , Macrófagos/microbiología , Células A549 , Adulto , Anciano , Burkholderia pseudomallei/patogenicidad , Línea Celular Tumoral , Femenino , Eliminación de Gen , Células HeLa , Humanos , Masculino , Melioidosis/microbiología , Melioidosis/patología , Técnicas Microbiológicas , Persona de Mediana Edad , Estudios Prospectivos , Secuenciación Completa del Genoma , Adulto JovenRESUMEN
BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease in tropical regions. It is necessary to understand the risk of acquiring this infection from the environment. METHODOLOGY /PRINCIPAL FINDINGS: The prevalence, concentration and genetic diversity of B. pseudomallei isolates collected from two sites in Buriram, Northeast Thailand were investigated. Forty-four environmental samples (18 from soil, 14 from rice rhizosphere, and 12 from water) were collected; of those 44 samples, 19 were collected from near a patient's residence and 25 from suspected exposure sites and compared with 10 clinical isolates of the patient. Quantitative culture was performed, and B. pseudomallei was identified using the latex agglutination test and matrix-laser absorption ionisation mass spectrometry. Genotyping was performed in 162 colonies from clinical (N = 10) and environmental samples (N = 152) using pulse-field gel electrophoresis (PFGE) followed by multi-locus sequence typing (MLST) of the clinical strain. B. pseudomallei was detected in 11 of the 44 environmental samples (1 from soil, 4 from rice rhizosphere, and 6 from water). The bacterial count in the positive soil sample was 115 CFU/g. The mean concentrations ± SDs of B. pseudomallei in the positive water and rhizosphere samples were 5.1 ± 5.5 CFU/ml and 80 ± 49 CFU/g, respectively. Six water samples with positive results were collected from a pond and water sources for drinking and daily use. All colonies isolated from the patient shared the same PFGE type (PT) indicating monoclonal infection of ST99. Although the 152 colonies from environmental isolates exhibited 25 PTs, none were identical to the patient's isolates. PT5 and PT7 were most common genotype among the environmental samples. CONCLUSIONS/SIGNIFICANCE: Diverse genotypes of B. pseudomallei were prevalent in the environment. However, the patient may have been infected with a low-density genotype. Intervention strategies for preventing B. pseudomallei infection are required.
Asunto(s)
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/aislamiento & purificación , Variación Genética , Melioidosis/microbiología , Monitoreo del Ambiente , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Tipificación de Secuencias Multilocus , Oryza/microbiología , Prevalencia , Microbiología del Suelo , Tailandia , Microbiología del AguaRESUMEN
Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei that results in high mortality. Hemolysin co-regulated protein 1 (Hcp1) and O-polysaccharide (OPS) are vaccine candidates and potential diagnostic antigens. The correlation of classes/subclasses of antibodies against these antigens with clinical characteristics of melioidosis patients is unknown. Antibodies in plasma samples from melioidosis patients and healthy donors were quantified by ELISA and compared with clinical features. In melioidosis patients, Hcp1 induced high IgG levels. OPS induced high IgG and IgA levels. The area under receiver operating characteristic curve (AUROCC) to discriminate melioidosis cases from healthy donors was highest for anti-Hcp1 IgG (0.92) compared to anti-Hcp1 IgA or IgM. In contrast, AUROCC for anti-OPS for IgG (0.91) and IgA (0.92) were comparable. Anti-Hcp1 IgG1 and anti-OPS IgG2 had the greatest AUROCCs (0.87 and 0.95, respectively) compared to other IgG subclasses for each antigen. Survivors had significantly higher anti-Hcp1 IgG3 levels than non-survivors. Male melioidosis patients with diabetes had higher anti-OPS IgA levels than males without diabetes. Thus, diverse and specific antibody responses are associated with distinct clinical characteristics in melioidosis, confirming the diagnostic utility of these responses and providing new insights into immune mechanisms.