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1.
Medicine (Baltimore) ; 100(25): e26160, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160382

RESUMO

ABSTRACT: Melioidosis is an infectious disease that is initiated by a bacteria recognized as Burkholderia pseudomallei. Despite the high fatality rate from melioidosis, there is a minimal published study about the disease in Malaysia.This study aimed to identify the prognostic factors of mortality among melioidosis patients in northern Malaysia.All inpatient patients who were admitted to Hospital Sultanah Bahiyah, Kedah and Hospital Tuanku Fauziah, Perlis with culture-confirmed melioidosis during the period 2014 to 2017 were included in the study. The study retrospectively collected 510 melioidosis patients from the Melioidosis Registry. Hazard ratio (HR) used in advanced multiple Cox regression was used to obtain the final model of prognostic factors of melioidosis. The analysis was performed using STATA/SE 14.0 for Windows software.From the results, among the admitted patients, 50.1% died at the hospital. The mean age for those who died was 55 years old, and they were mostly male. The most common underlying disease was diabetes mellitus (69.8%), followed by hypertension (32.7%). The majority of cases (86.8%) were bacteremic. The final Cox model identified 5 prognostic factors of mortality among melioidosis patients. The factors were diabetes mellitus, type of melioidosis, platelet count, white blood cell count, and urea value. The results showed that bacteremic melioidosis increased the risk of dying by 3.47 (HR: 3.47, 95% confidence intervals [CI]: 1.67-7.23, P = .001) compared to non-bacteremic melioidosis. Based on the blood investigations, the adjusted HRs from the final model showed that all 3 blood investigations were included as the prognostic factors for the disease (low platelet: HR = 1.76, 95% CI: 1.22-2.54, P = .003; high white blood cell: HR = 1.49, 95% CI 1.06-2.11, P = .023; high urea: HR = 2.92, 95% CI: 1.76-4.85, P < .001; and low level of urea: HR = 2.69, 95% CI: 1.69-4.29, P < .001). By contrast, melioidosis patients with diabetic had 30.0% lower risk of dying from melioidosis compared to those with non-diabetic (HR = 0.70, 95% CI: 0.52-0.94, P = .016).Identifying the prognostic factors of mortality in patients with melioidosis allows a guideline of early management in these patients, which may improve patient's survival.


Assuntos
Antibacterianos/uso terapêutico , Burkholderia pseudomallei/isolamento & purificação , Diabetes Mellitus/epidemiologia , Hipertensão/epidemiologia , Melioidose/mortalidade , Adulto , Idoso , Feminino , Mortalidade Hospitalar , Humanos , Contagem de Leucócitos , Malásia/epidemiologia , Masculino , Melioidose/sangue , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Pessoa de Meia-Idade , Contagem de Plaquetas , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Proteção , Sistema de Registros/estatística & dados numéricos , Estudos Retrospectivos , Medição de Risco/métodos , Fatores de Risco , Ureia/sangue
2.
BMC Microbiol ; 21(1): 137, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947331

RESUMO

BACKGROUND: Burkholderia pseudomallei, a facultative intracellular bacterium, is the aetiological agent of melioidosis that is responsible for up to 40% sepsis-related mortality in epidemic areas. However, no effective vaccine is available currently, and the drug resistance is also a major problem in the treatment of melioidosis. Therefore, finding new clinical treatment strategies in melioidosis is extremely urgent. RESULTS: We demonstrated that tauroursodeoxycholic acid (TUDCA), a clinically available endoplasmic reticulum (ER) stress inhibitor, can promote B. pseudomallei clearance both in vivo and in vitro. In this study, we investigated the effects of TUDCA on the survival of melioidosis mice, and found that treatment with TUDCA significantly decreased intracellular survival of B. pseudomallei. Mechanistically, we found that B. pseudomallei induced apoptosis and activated IRE1 and PERK signaling ways of ER stress in RAW264.7 macrophages. TUDCA treatment could reduce B. pseudomallei-induced ER stress in vitro, and TUDCA is protective in vivo. CONCLUSION: Taken together, our study has demonstrated that B. pseudomallei infection results in ER stress-induced apoptosis, and TUDCA enhances the clearance of B. pseudomallei by inhibiting ER stress-induced apoptosis both in vivo and in vitro, suggesting that TUDCA could be used as a potentially alternative treatment for melioidosis.


Assuntos
Burkholderia pseudomallei/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melioidose/microbiologia , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Burkholderia pseudomallei/efeitos dos fármacos , Linhagem Celular , Melioidose/tratamento farmacológico , Camundongos , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Ácido Tauroquenodesoxicólico/uso terapêutico
3.
Sci Rep ; 11(1): 10405, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001967

RESUMO

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.


Assuntos
Proteínas de Bactérias/genética , Burkholderia pseudomallei/patogenicidade , Regulação Bacteriana da Expressão Gênica , Melioidose/microbiologia , Proteínas Repressoras/genética , Animais , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/genética , Sequenciamento de Cromatina por Imunoprecipitação , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Células RAW 264.7 , RNA-Seq , Regulon , Proteínas Repressoras/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
4.
PLoS Pathog ; 17(5): e1009604, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34048488

RESUMO

Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/genética , Melioidose/microbiologia , Adaptação Fisiológica , Anaerobiose , Animais , Proteínas de Bactérias/genética , Burkholderia pseudomallei/patogenicidade , Burkholderia pseudomallei/fisiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Hipóxia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Nitratos/metabolismo , Oxirredução , Transcriptoma , Virulência
5.
Appl Environ Microbiol ; 87(12): e0301920, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33811022

RESUMO

Melioidosis is a life-threatening disease in humans caused by the Gram-negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 h, a rapid diagnosis of melioidosis is critical for ensuring that an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and the whole genome was sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an area of melioidosis endemicity over 11 months. The 94TF-LAA took less than 5 min to produce positive agglutination, demonstrating 98% (95% confidence interval [CI] of 94.2% to 99.59%) sensitivity and 83% (95% CI of 75.64% to 88.35%) specificity compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen. IMPORTANCE Rapid diagnosis of melioidosis is essential for ensuring that optimal antibiotic courses are prescribed to patients and thus warrants the development of cost-effective and easy-to-use tests for implementation in underresourced areas such as northeastern Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust and easy to manufacture, with many tail fibers (such as 94TF investigated here) capable of targeting a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many areas where melioidosis is endemic.


Assuntos
Bacteriófagos , Burkholderia pseudomallei/virologia , Melioidose/diagnóstico , Burkholderia pseudomallei/genética , Proteínas do Capsídeo , Humanos , Testes de Fixação do Látex , Melioidose/microbiologia , Sensibilidade e Especificidade
6.
Molecules ; 26(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672903

RESUMO

Burkholderia pseudomallei is the causative pathogen of melioidosis and this bacterium is resistant to several antibiotics. Silver nanoparticles (AgNPs) are an interesting agent to develop to solve this bacterial resistance. Here, we characterize and assess the antimelioidosis activity of AgNPs against these pathogenic bacteria. AgNPs were characterized and displayed a maximum absorption band at 420 nm with a spherical shape, being well-monodispersed and having high stability in solution. The average size of AgNPs is 7.99 ± 1.46 nm. The antibacterial efficacy of AgNPs was evaluated by broth microdilution. The bactericidal effect of AgNPs was further assessed by time-kill kinetics assay. Moreover, the effect of AgNPs on the inhibition of the established biofilm was investigated by the crystal violet method. In parallel, a study of the resistance induction development of B. pseudomallei towards AgNPs with efflux pump inhibiting effect was performed. We first found that AgNPs had strong antibacterial activity against both susceptible and ceftazidime-resistant (CAZ-resistant) strains, as well as being efficiently active against B. pseudomallei CAZ-resistant strains with a fast-killing mode via a bactericidal effect within 30 min. These AgNPs did not only kill planktonic bacteria in broth conditions, but also in established biofilm. Our findings first documented that the resistance development was not induced in B. pseudomallei toward AgNPs in the 30th passage. We found that AgNPs still showed an effective efflux pump inhibiting effect against these bacteria after prolonged exposure to AgNPs at sublethal concentrations. Thus, AgNPs have valuable properties for being a potent antimicrobial agent to solve the antibiotic resistance problem in pathogens.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Burkholderia pseudomallei/fisiologia , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Nanopartículas Metálicas/uso terapêutico , Prata/uso terapêutico , Taninos/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/efeitos dos fármacos , Ceftazidima/farmacologia , Ceftazidima/uso terapêutico , Farmacorresistência Bacteriana/efeitos dos fármacos , Difusão Dinâmica da Luz , Cinética , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Fenótipo , Prata/farmacologia , Eletricidade Estática , Taninos/farmacologia
7.
Nat Commun ; 12(1): 1907, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772012

RESUMO

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.


Assuntos
Proteínas de Bactérias/genética , Burkholderia pseudomallei/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Genes Bacterianos/genética , Fatores de Virulência/genética , Animais , Burkholderia pseudomallei/citologia , Burkholderia pseudomallei/patogenicidade , Linhagem Celular Tumoral , Membrana Celular/microbiologia , Citoplasma/microbiologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Melioidose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Análise de Célula Única/métodos , Vacúolos/microbiologia , Virulência/genética
8.
PLoS Negl Trop Dis ; 15(2): e0008913, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33592059

RESUMO

BACKGROUND: Melioidosis is an endemic disease in southeast Asia and northern Australia caused by the saprophytic bacteria Burkholderia pseudomallei, with a high mortality rate. The clinical presentation is multifaceted, with symptoms ranging from acute septicemia to multiple chronic abscesses. Here, we report a chronic case of melioidosis in a patient who lived in Malaysia in the 70s and was suspected of contracting tuberculosis. Approximately 40 years later, in 2014, he was diagnosed with pauci-symptomatic melioidosis during a routine examination. Four strains were isolated from a single sample. They showed divergent morphotypes and divergent antibiotic susceptibility, with some strains showing resistance to trimethoprim-sulfamethoxazole and fluoroquinolones. In 2016, clinical samples were still positive for B. pseudomallei, and only one type of strain, showing atypical resistance to meropenem, was isolated. PRINCIPAL FINDINGS: We performed whole genome sequencing and RT-qPCR analysis on the strains isolated during this study to gain further insights into their differences. We thus identified two types of resistance mechanisms in these clinical strains. The first one was an adaptive and transient mechanism that disappeared during the course of laboratory sub-cultures; the second was a mutation in the efflux pump regulator amrR, associated with the overexpression of the related transporter. CONCLUSION: The development of such mechanisms may have a clinical impact on antibiotic treatment. Indeed, their transient nature could lead to an undiagnosed resistance. Efflux overexpression due to mutation leads to an important multiple resistance, reducing the effectiveness of antibiotics during treatment.


Assuntos
Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/genética , Farmacorresistência Bacteriana Múltipla/genética , Melioidose/microbiologia , Idoso de 80 Anos ou mais , Antibacterianos , Humanos , Malásia , Masculino , Proteínas de Membrana Transportadoras/genética , Meropeném , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real , Combinação Trimetoprima e Sulfametoxazol , Sequenciamento Completo do Genoma
9.
PLoS Negl Trop Dis ; 15(2): e0009016, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33617546

RESUMO

Burkholderia pseudomallei is the causative agent of melioidosis, which is a Gram negative, facultative intracellular bacterium. Disease is prevalent in SE Asia and in northern Australia, as well as in other tropical and subtropical regions. Recently, there is an increasing awareness of the importance of bacterial ingestion as a potential route of infection, particularly in cases of unexplained origin of the disease. The marmoset is a New World Monkey (NWM) species that is being developed as an alternative NHP model to complement the more traditionally used Old World Monkeys (OWM). Models have been developed for the traditional routes of disease acquisition, subcutaneous and inhalational. This manuscript details the development and characterisation of an ingestion model of melioidosis. Dose-ranging study assessed the lethality of B. pseudomallei and disease progression was assessed by euthanizing animals at predetermined time points, 12, 36, 48 and 54 hours post-challenge. Challenge doses of greater than 6.2 x 106 cfu resulted in an acute, lethal, febrile disease. Following challenge the lung was the first organ, outside of the gastrointestinal tract, to become colonised. Enteritis (duodenitis, ileitis and/or jejunitis) was observed in sections of the small intestine from animals that succumbed to disease. However, the most severe pathological features were observed in the mesenteric lymph nodes from these animals. These findings are consistent with lymphatic draining as route of dissemination.


Assuntos
Burkholderia pseudomallei/fisiologia , Sistema Linfático/microbiologia , Melioidose/patologia , Animais , Burkholderia pseudomallei/patogenicidade , Callithrix , Modelos Animais de Doenças , Enterite/microbiologia , Feminino , Pulmão/microbiologia , Linfonodos/microbiologia , Linfonodos/patologia , Masculino , Melioidose/microbiologia
10.
mSphere ; 6(1)2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536328

RESUMO

Burkholderia pseudomallei is a Gram-negative bacterium that causes the sapronotic disease melioidosis. An outbreak in 2003 in the state of Ceara, Brazil, resulted in subsequent surveillance and environmental sampling which led to the recognition of B. pseudomallei as an endemic pathogen in that area. From 2003 to 2015, 24 clinical and 12 environmental isolates were collected across Ceara along with one from the state of Alagoas. Using next-generation sequencing, multilocus sequence typing, and single nucleotide polymorphism analysis, we characterized the genomic diversity of this collection to better understand the population structure of B. pseudomallei associated with Ceara. We found that the isolates in this collection form a distinct subclade compared to other examples from the Western Hemisphere. Substantial genetic diversity among the clinical and environmental isolates was observed, with 14 sequence types (STs) identified among the 37 isolates. Of the 31,594 core single-nucleotide polymorphisms (SNPs) identified, a high proportion (59%) were due to recombination. Because recombination events do not follow a molecular clock, the observation of high occurrence underscores the importance of identifying and removing recombination SNPs prior to evolutionary reconstructions and inferences in public health responses to B. pseudomallei outbreaks. Our results suggest long-term B. pseudomallei prevalence in this recently recognized region of melioidosis endemicity.IMPORTANCE B. pseudomallei causes significant morbidity and mortality, but its geographic prevalence and genetic diversity are not well characterized, especially in the Western Hemisphere. A better understanding of the genetic relationships among clinical and environmental isolates will improve knowledge of the population structure of this bacterium as well as the ability to conduct epidemiological investigations of cases of melioidosis.


Assuntos
Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/genética , Variação Genética , Genoma Bacteriano , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , DNA Bacteriano/genética , Surtos de Doenças/estatística & dados numéricos , Genômica/métodos , Genótipo , Humanos , Masculino , Melioidose/epidemiologia , Melioidose/microbiologia , Filogenia , Análise de Sequência de DNA
11.
Trop Med Int Health ; 26(6): 664-671, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33590932

RESUMO

OBJECTIVES: Melioidosis, caused by Burkholderia pseudomallei, is prevalent in rural areas of Malaysia. The aim of this study is to delineate the epidemiology and predictors of mortality from melioidosis in Kapit district, Sarawak. METHODS: For this retrospective study of patients with culture-confirmed melioidosis admitted to Kapit Hospital, Sarawak, Malaysia, between July 2016 and July 2019, epidemiological, clinical and microbiological data were obtained. Univariate and multivariate logistic regression analyses were used to determine predictors of mortality. RESULTS: Seventy three patients met inclusion criteria. Diabetes mellitus (28.8%) and hypertension (27.4%) were primary co-morbidities. Clinical spectrum of melioidosis ranged from bacteraemia (64.4%), pneumonia (61.6%) and internal organ abscesses (49.3%) to localised soft tissue (21.9%) and joint abscesses (6.9%). Mortality rate was 12.3%. Bacteraemia and pneumonia were significantly associated with septic shock, whereas patients with soft tissue abscesses tended to present with a milder form of melioidosis without septic shock. Septic shock, mechanical ventilation, intensive care unit admission, serum urea, creatinine, bicarbonate, albumin and aspartate transaminase were all significantly associated with increased mortality on univariate analysis (all P < 0.05). Multivariate analysis revealed that low serum bicarbonate (P = 0.004, OR 0.64, 95% CI 0.48-0.87) and albumin (P = 0.031, OR 0.73, 95% CI 0.54-0.97) could be associated with a higher mortality. CONCLUSION: Melioidosis remains a fatal infection and commonly presents with septic shock, in the form of bacteraemia and pneumonia. Two routine clinical parameters, serum bicarbonate and serum albumin, may have important prognostic implications in septicaemic melioidosis.


Assuntos
Melioidose/complicações , Melioidose/epidemiologia , Melioidose/microbiologia , Melioidose/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/etiologia , Bicarbonatos/sangue , Biomarcadores/sangue , Burkholderia pseudomallei/isolamento & purificação , Comorbidade , Feminino , Humanos , Malásia/epidemiologia , Masculino , Pessoa de Meia-Idade , Pneumonia/etiologia , Estudos Retrospectivos , Albumina Sérica , Choque Séptico/etiologia , Adulto Jovem
12.
PLoS Negl Trop Dis ; 15(2): e0009125, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33571211

RESUMO

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1ß, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1ß at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis.


Assuntos
Aerossóis , Burkholderia pseudomallei , Melioidose/microbiologia , Melioidose/patologia , Animais , Ásia Sudeste , Austrália , Bacteriemia , Medula Óssea/patologia , Quimiocinas/metabolismo , Chlorocebus aethiops , Citocinas , Modelos Animais de Doenças , Progressão da Doença , Humanos , Fígado/patologia , Pulmão/patologia , Macaca mulatta , Baço/patologia , Telemetria , Tailândia , Virulência
13.
Microb Genom ; 7(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33565959

RESUMO

Burkholderia pseudomallei, a soil-dwelling Gram-negative bacterium, is the causative agent of the endemic tropical disease melioidosis. Clinical manifestations of B. pseudomallei infection range from acute or chronic localized infection in a single organ to fulminant septicaemia in multiple organs. The diverse clinical manifestations are attributed to various factors, including the genome plasticity across B. pseudomallei strains. We previously characterized B. pseudomallei strains isolated in Malaysia and noted different levels of virulence in model hosts. We hypothesized that the difference in virulence might be a result of variance at the genome level. In this study, we sequenced and assembled four Malaysian clinical B. pseudomallei isolates, UKMR15, UKMPMC2000, UKMD286 and UKMH10. Phylogenomic analysis showed that Malaysian subclades emerged from the Asian subclade, suggesting that the Malaysian strains originated from the Asian region. Interestingly, the low-virulence strain, UKMH10, was the most distantly related compared to the other Malaysian isolates. Genomic island (GI) prediction analysis identified a new island of 23 kb, GI9c, which is present in B. pseudomallei and Burkholderia mallei, but not Burkholderia thailandensis. Genes encoding known B. pseudomallei virulence factors were present across all four genomes, but comparative analysis of the total gene content across the Malaysian strains identified 104 genes that are absent in UKMH10. We propose that these genes may encode novel virulence factors, which may explain the reduced virulence of this strain. Further investigation on the identity and role of these 104 proteins may aid in understanding B. pseudomallei pathogenicity to guide the design of new therapeutics for treating melioidosis.


Assuntos
Burkholderia pseudomallei/classificação , Melioidose/microbiologia , Tipagem de Sequências Multilocus/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Tipagem Bacteriana , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Burkholderia pseudomallei/patogenicidade , Tamanho do Genoma , Genoma Bacteriano , Ilhas Genômicas , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Malásia , Filogenia , Fatores de Virulência/genética
15.
Future Microbiol ; 16: 271-288, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33595347

RESUMO

Melioidosis is an emerging infectious disease with an estimated global burden of 4.64 million disability-adjusted life years per year. A major determinant related to poor disease outcomes is delay to diagnosis due to the fact that identification of the causative agent Burkholderia pseudomallei may be challenging. Over the last 25 years, advances in molecular diagnostic techniques have resulted in the potential for rapid and accurate organism detection and identification direct from clinical samples. While these methods are not yet routine in clinical practice, laboratory diagnosis of infectious diseases is transitioning to culture-independent techniques. This review article aims to evaluate molecular methods for melioidosis diagnosis direct from clinical samples and discuss current and future utility and limitations.


Assuntos
Burkholderia pseudomallei/genética , Melioidose/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Animais , Burkholderia pseudomallei/isolamento & purificação , Burkholderia pseudomallei/fisiologia , DNA Bacteriano/genética , Humanos , Melioidose/microbiologia , Reação em Cadeia da Polimerase em Tempo Real
16.
EBioMedicine ; 63: 103152, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33285499

RESUMO

BACKGROUND: Antimicrobial resistance (AMR) poses a major threat to human health. Whole-genome sequencing holds great potential for AMR identification; however, there remain major gaps in accurately and comprehensively detecting AMR across the spectrum of AMR-conferring determinants and pathogens. METHODS: Using 16 wild-type Burkholderia pseudomallei and 25 with acquired AMR, we first assessed the performance of existing AMR software (ARIBA, CARD, ResFinder, and AMRFinderPlus) for detecting clinically relevant AMR in this pathogen. B. pseudomallei was chosen due to limited treatment options, high fatality rate, and AMR caused exclusively by chromosomal mutation (i.e. single-nucleotide polymorphisms [SNPs], insertions-deletions [indels], copy-number variations [CNVs], inversions, and functional gene loss). Due to poor performance with existing tools, we developed ARDaP (Antimicrobial Resistance Detection and Prediction) to identify the spectrum of AMR-conferring determinants in B. pseudomallei. FINDINGS: CARD, ResFinder, and AMRFinderPlus failed to identify any clinically-relevant AMR in B. pseudomallei; ARIBA identified AMR encoded by SNPs and indels that were manually added to its database. However, none of these tools identified CNV, inversion, or gene loss determinants, and ARIBA could not differentiate AMR determinants from natural genetic variation. In contrast, ARDaP accurately detected all SNP, indel, CNV, inversion, and gene loss AMR determinants described in B. pseudomallei (n≈50). Additionally, ARDaP accurately predicted three previously undescribed determinants. In mixed strain data, ARDaP identified AMR to as low as ~5% allelic frequency. INTERPRETATION: Existing AMR software packages are inadequate for chromosomal AMR detection due to an inability to detect resistance conferred by CNVs, inversions, and functional gene loss. ARDaP overcomes these major shortcomings. Further, ARDaP enables AMR prediction from mixed sequence data down to 5% allelic frequency, and can differentiate natural genetic variation from AMR determinants. ARDaP databases can be constructed for any microbial species of interest for comprehensive AMR detection. FUNDING: National Health and Medical Research Council (BJC, EPP, DSS); Australian Government (DEM, ES); Advance Queensland (EPP, DSS).


Assuntos
Burkholderia pseudomallei/efeitos dos fármacos , Melioidose/microbiologia , Antibacterianos/farmacologia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Biologia Computacional/métodos , Farmacorresistência Bacteriana , Genes Bacterianos , Genoma Bacteriano , Genômica/métodos , Humanos , Melioidose/tratamento farmacológico , Testes de Sensibilidade Microbiana , Mutação , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
18.
BMJ Case Rep ; 13(12)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33370949

RESUMO

Melioidosis is caused by the tropical soil pathogen Burkholderia pseudomallei Infection, usually in the form of pneumonia, disproportionately affects people with a risk factor for immune dysregulation and mortality remains high even with treatment. Climate change and increasing rates of diabetes render the populations of endemic areas increasingly vulnerable to the disease, which is emerging as a serious global health threat. We present here a case of a 68-year-old man from northern Australia with sepsis and osteoarticular melioidosis of the hip, and explore the links between diabetes mellitus and melioidosis, particularly with respect to musculoskeletal infection.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Diabetes Mellitus Tipo 2/complicações , Melioidose/diagnóstico , Osteoartrite do Quadril/microbiologia , Sepse/microbiologia , Idoso , Antibacterianos/uso terapêutico , Austrália , Burkholderia pseudomallei/imunologia , Diabetes Mellitus Tipo 2/imunologia , Quimioterapia Combinada/métodos , Articulação do Quadril/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Masculino , Melioidose/tratamento farmacológico , Melioidose/imunologia , Melioidose/microbiologia , Osteoartrite do Quadril/diagnóstico , Osteoartrite do Quadril/tratamento farmacológico , Osteoartrite do Quadril/imunologia , Sepse/diagnóstico , Sepse/tratamento farmacológico , Sepse/imunologia , Resultado do Tratamento
19.
BMJ Case Rep ; 13(11)2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33148566

RESUMO

A 48-year-old man who worked in mining in remote, northern Australia was transferred from a rural hospital 5 days after the onset of headaches, subjective fevers and flaccid paralysis of the left upper limb. Initial investigations demonstrated inflammatory cerebrospinal fluid (CSF) changes and a longitudinally extensive cervical cord lesion. Given two serial negative blood and CSF cultures, he was treated as inflammatory myelitis with intravenous methylprednisolone. Despite the initial improvement in pain and left arm power, the patient's neurological deficit plateaued and then deteriorated with worsening neck pain, diaphragmatic dysfunction and dysphagia requiring intubation and respiratory support. A third CSF culture isolated Burkholderia pseudomallei confirming a diagnosis of neuro-melioidosis. Repeat imaging revealed the rostral extension of the original spinal cord lesion into the medulla and pons. Over the next 4 weeks, the patient's neurological deficits slowly improved with continued intravenous antibiotic therapy with meropenem and oral trimethoprim/sulfamethoxazole.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Infecções do Sistema Nervoso Central/diagnóstico , Melioidose/diagnóstico , Doenças Raras , Infecções do Sistema Nervoso Central/líquido cefalorraquidiano , Infecções do Sistema Nervoso Central/microbiologia , Líquido Cefalorraquidiano/microbiologia , Vértebras Cervicais , Diagnóstico Diferencial , Humanos , Imageamento por Ressonância Magnética , Masculino , Melioidose/líquido cefalorraquidiano , Melioidose/microbiologia , Pessoa de Meia-Idade
20.
Sci Rep ; 10(1): 17972, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087788

RESUMO

Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.


Assuntos
Burkholderia pseudomallei/patogenicidade , Fusão Celular , Melioidose/microbiologia , Fagócitos/fisiologia , Tetraspaninas/fisiologia , Células A549 , Anticorpos Monoclonais , Sistemas CRISPR-Cas , Células Cultivadas , Células Gigantes/microbiologia , Humanos , Melioidose/terapia , Tetraspanina 28 , Tetraspanina 29 , Tetraspaninas/metabolismo
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