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1.
BMC Infect Dis ; 19(1): 984, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752745

RESUMO

BACKGROUND: Burkholderia pseudomallei is a gram-negative bacterium and the causative pathogen of melioidosis, which manifests a variety ranges of infection symptoms. However, deep venous thrombosis (DVT) and pulmonary embolism (PE) secondary to bacteremic melioidosis are rarely documented in the literature. Herein, we reported a fatal case of melioidosis combined with DVT and PE. CASE PRESENTATION: A 54-year-old male construction worker and farmer with a history of diabetes was febrile, painful in left thigh, swelling in left lower limb, with chest tightness and shortness of breath for 4 days. He was later diagnosed as DVT of left lower extremity and PE. The culture of his blood, sputum and bone marrow samples grew B. pseudomallei. The subject was administrated with antibiotics (levofloxacin, cefoperazone/tazobactam, and imipenem) according to antimicrobial susceptibility testing and low molecular heparin for venous thrombosis. However, even after appropriate treatment, the patient deteriorated rapidly, and died 2 weeks after admission. CONCLUSIONS: This study enhanced awareness of the risk of B. pseudomallei bloodstream infection in those with diabetes. If a patient has predisposing factors of melioidosis, when DVT is suspected, active investigation and multiple therapeutic interventions should be implemented immediately to reduce mortality rate.


Assuntos
Melioidose/complicações , Embolia Pulmonar/etiologia , Trombose Venosa/etiologia , Antibacterianos/administração & dosagem , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , China , Evolução Fatal , Heparina/administração & dosagem , Humanos , Masculino , Melioidose/microbiologia , Pessoa de Meia-Idade , Embolia Pulmonar/tratamento farmacológico , Trombose Venosa/tratamento farmacológico
2.
PLoS Negl Trop Dis ; 13(9): e0007672, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31487283

RESUMO

In the wet-dry tropics of Northern Australia, drinking water in remote communities is mostly sourced from bores accessing groundwater. Many aquifers contain naturally high levels of iron and some are shallow with surface water intrusion in the wet season. Therefore, environmental bacteria such as iron-cycling bacteria promoting biofilm formation in pipes or opportunistic pathogens can occur in these waters. An opportunistic pathogen endemic to northern Australia and Southeast Asia and emerging worldwide is Burkholderia pseudomallei. It causes the frequently fatal disease melioidosis in humans and animals. As we know very little about the microbial composition of drinking water in remote communities, this study aimed to provide a first snapshot of the microbiota and occurrence of opportunistic pathogens in bulk water and biofilms from the source and through the distribution system of three remote water supplies with varying iron levels. Using 16s-rRNA gene sequencing, we found that the geochemistry of the groundwater had a substantial impact on the untreated microbiota. Different iron-cycling bacteria reflected differences in redox status and nutrients. We cultured and sequenced B. pseudomallei from bores with elevated iron and from a multi-species biofilm which also contained iron-oxidizing Gallionella, nitrifying Nitrospira and amoebae. Gallionella are increasingly used in iron-removal filters in water supplies and more research is needed to examine these interactions. Similar to other opportunistic pathogens, B. pseudomallei occurred in water with low organic carbon levels and with low heterotrophic microbial growth. No B. pseudomallei were detected in treated water; however, abundant DNA of another opportunistic pathogen group, non-tuberculous mycobacteria was recovered from treated parts of one supply. Results from this study will inform future studies to ultimately improve management guidelines for water supplies in the wet-dry tropics.


Assuntos
Bactérias/isolamento & purificação , Água Potável/microbiologia , Austrália , Bactérias/classificação , Bactérias/genética , Biodiversidade , Biofilmes , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Burkholderia pseudomallei/fisiologia , Água Potável/química , Gallionellaceae/genética , Gallionellaceae/isolamento & purificação , Gallionellaceae/fisiologia , Ferro/análise , Filogenia , População Rural , Poluição da Água/análise , Abastecimento de Água
3.
PLoS Negl Trop Dis ; 13(9): e0007727, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31487287

RESUMO

BACKGROUND: Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated. METHODOLOGY/PRINCIPAL FINDINGS: We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp. CONCLUSIONS/SIGNIFICANCE: B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Burkholderia/classificação , Burkholderia/isolamento & purificação , Burkholderia pseudomallei/genética , Ilhas Genômicas , Melioidose , Filogenia , Reação em Cadeia da Polimerase/métodos , Porto Rico , Análise de Sequência de DNA , Microbiologia do Solo , Microbiologia da Água
4.
Nucleic Acids Res ; 47(17): 9448-9463, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31400118

RESUMO

Overcoming lysogenization defect (OLD) proteins constitute a family of uncharacterized nucleases present in bacteria, archaea, and some viruses. These enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst replication, recombination, and repair proteins. The in vivo activities of OLD proteins remain poorly understood and no definitive structural information exists. Here we identify and define two classes of OLD proteins based on differences in gene neighborhood and amino acid sequence conservation and present the crystal structures of the catalytic C-terminal regions from the Burkholderia pseudomallei and Xanthamonas campestris p.v. campestris Class 2 OLD proteins at 2.24 Å and 1.86 Å resolution respectively. The structures reveal a two-domain architecture containing a Toprim domain with altered architecture and a unique helical domain. Conserved side chains contributed by both domains coordinate two bound magnesium ions in the active site of B. pseudomallei OLD in a geometry that supports a two-metal catalysis mechanism for cleavage. The spatial organization of these domains additionally suggests a novel mode of DNA binding that is distinct from other Toprim containing proteins. Together, these findings define the fundamental structural properties of the OLD family catalytic core and the underlying mechanism controlling nuclease activity.


Assuntos
Burkholderia pseudomallei/química , Domínio Catalítico/genética , Desoxirribonucleases/ultraestrutura , Conformação Proteica , Xanthomonas/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos/genética , Burkholderia pseudomallei/genética , Catálise , Desoxirribonucleases/química , Desoxirribonucleases/genética , Evolução Molecular , Lisogenia/genética , Metais/química , Domínios Proteicos/genética , Alinhamento de Sequência , Xanthomonas/genética
5.
Microbiol Res ; 226: 48-54, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284944

RESUMO

The Burkholderia pseudomallei complex consists of six phylogenetically related Gram-negative bacterial species that include environmental saprophytes and mammalian pathogens. These microbes possess multiple type VI secretion systems (T6SS) that provide a fitness advantage in diverse niches by translocating effector molecules into prokaryotic and eukaryotic cells in a contact-dependent manner. Several recent studies have elucidated the regulation and function of T6SS-2, a novel contact-independent member of the T6SS family. Expression of the T6SS-2 gene cluster is repressed by OxyR, Zur and TctR and is activated by GvmR and reactive oxygen species (ROS). The last two genes of the T6SS-2 gene cluster encode a zincophore (TseZ) and a manganeseophore (TseM) that are exported into the extracellular milieu in a contact-independent fashion when microbes encounter oxidative stress. TseZ and TseM bind Zn2+ and Mn2+, respectively, and deliver them to bacteria where they provide protection against the lethal effects of ROS. The TonB-dependent transporters that interact with TseZ and TseM, and actively transport Zn2+ and Mn2+ across the outer membrane, have also been identified. Finally, T6SS-2 provides a contact-independent growth advantage in nutrient limited environments and is critical for virulence in Galleria mellonella larvae, but is dispensable for virulence in rodent models of infection.


Assuntos
Proteínas de Bactérias/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Manganês/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Zinco/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/classificação , Regulação Bacteriana da Expressão Gênica , Genes Reguladores/genética , Homeostase , Larva , Proteínas de Membrana Transportadoras/genética , Metiltransferases , Família Multigênica , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Virulência/genética
6.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31331957

RESUMO

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomallei ΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.


Assuntos
Proteínas de Bactérias/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidade , Ciclofilinas/genética , Melioidose/microbiologia , Proteoma/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/metabolismo , Linhagem Celular , Ciclofilinas/deficiência , Feminino , Deleção de Genes , Expressão Gênica , Homeostase/genética , Macrófagos/microbiologia , Melioidose/tratamento farmacológico , Melioidose/mortalidade , Melioidose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos dos fármacos , Proteoma/classificação , Proteoma/metabolismo , Análise de Sobrevida , Virulência
7.
Biomed Res Int ; 2019: 9451791, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31355287

RESUMO

Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.


Assuntos
Burkholderia pseudomallei/genética , DNA Bacteriano/genética , Leptospira/genética , Leptospirose , Melioidose , Reação em Cadeia da Polimerase em Tempo Real , Animais , Humanos , Hidrólise , Leptospirose/diagnóstico , Leptospirose/genética , Melioidose/diagnóstico , Melioidose/genética
8.
PLoS Negl Trop Dis ; 13(7): e0007369, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31348781

RESUMO

The Tier 1 select agent Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a high mortality disease. Variably present genetic markers used to elucidate strain origin, relatedness and virulence in B. pseudomallei include the Burkholderia intracellular motility factor A (bimA) and filamentous hemagglutinin 3 (fhaB3) gene variants. Three lipopolysaccharide (LPS) O-antigen types in B. pseudomallei have been described, which vary in proportion between Australian and Asian isolates. However, it remains unknown if these LPS types can be used as genetic markers for geospatial analysis within a contiguous melioidosis-endemic region. Using a combination of whole-genome sequencing (WGS), statistical analysis and geographical mapping, we examined if the LPS types can be used as geographical markers in the Northern Territory, Australia. The clinical isolates revealed that LPS A prevalence was highest in the Darwin and surrounds (n = 660; 96% being LPS A and 4% LPS B) and LPS B in the Katherine and Katherine remote and East Arnhem regions (n = 79; 60% being LPS A and 40% LPS B). Bivariate logistics regression of 999 clinical B. pseudomallei isolates revealed that the odds of getting a clinical isolate with LPS B was highest in East Arnhem in comparison to Darwin and surrounds (OR 19.5, 95% CI 9.1-42.0; p<0.001). This geospatial correlation was subsequently confirmed by geographically mapping the LPS type from 340 environmental Top End strains. We also found that in the Top End, the minority bimA genotype bimABm has a similar remote region geographical footprint to that of LPS B. In addition, correlation of LPS type with multi-locus sequence typing (MLST) was strong, and where multiple LPS types were identified within a single sequence type, WGS confirmed homoplasy of the MLST loci. The clinical, sero-diagnostic and vaccine implications of geographically-based B. pseudomallei LPS types, and their relationships to regional and global dispersal of melioidosis, require global collaborations with further analysis of larger clinically and geospatially-linked datasets.


Assuntos
Burkholderia pseudomallei/genética , DNA Bacteriano/genética , Lipopolissacarídeos/genética , Técnicas de Tipagem Bacteriana , Burkholderia pseudomallei/classificação , Microbiologia Ambiental , Marcadores Genéticos , Variação Genética , Genoma Bacteriano , Genótipo , Humanos , Melioidose/epidemiologia , Melioidose/microbiologia , Tipagem de Sequências Multilocus , Northern Territory , Antígenos O/genética , Filogenia , Análise de Sequência de DNA , Clima Tropical , Virulência , Sequenciamento Completo do Genoma
9.
PLoS One ; 14(7): e0213416, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31283772

RESUMO

Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , DNA Bacteriano/análise , Melioidose/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Tipagem Bacteriana/economia , Técnicas de Tipagem Bacteriana/métodos , Burkholderia pseudomallei/genética , Primers do DNA/genética , DNA Bacteriano/genética , Humanos , Limite de Detecção , Melioidose/sangue , Melioidose/microbiologia , Técnicas de Amplificação de Ácido Nucleico/economia , Recombinases/química , Microbiologia do Solo , Fatores de Tempo
10.
Emerg Infect Dis ; 25(8)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31310232

RESUMO

We report 7 cases of melioidosis in Colombia and comparision of 4 commercial systems for identifying Burkholderia pseudomallei. Phoenix systems were not a definitive method for identifying B. pseudomallei. For accurate identification, we recommend including this bacterium in the library databases of matrix-assisted laser desorption/ionization mass spectrometry systems in Latin America.


Assuntos
Burkholderia pseudomallei , Melioidose/diagnóstico , Melioidose/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Colômbia , DNA Espaçador Ribossômico , Humanos , Melioidose/tratamento farmacológico , Testes de Sensibilidade Microbiana , Técnicas de Diagnóstico Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Resultado do Tratamento
11.
PLoS Negl Trop Dis ; 13(5): e0007312, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31091290

RESUMO

BACKGROUND: Melioidosis is gaining recognition as an emerging infectious disease with diverse clinical manifestations and high-case fatality rates worldwide. However, the molecular epidemiology of the disease outside the endemic regions such as northeast part of Thailand and northern Australia remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Clinical data and B. pseudomallei isolates obtained from 199 culture-confirmed cases of melioidosis diagnosed during 2006-2016 in South India were used to elucidate the host and pathogen specific variable virulence determinants associated with clinical presentations and disease outcome. Further, we determined the temporal variations and the influence of ecological factors on B.pseudomallei Lipopolysaccharide (LPS) genotypes causing infections. Severe forms of the disease were observed amongst 169 (85%) patients. Renal dysfunction and infection due to B.pseudomallei harboring BimABm variant had significant associations with severe forms of the disease. Diabetes mellitus, septicemic melioidosis and infection due to LPSB genotype were independent risk factors for mortality. LPSB (74%) and LPSA (20.6%) were the prevalent genotypes causing infections. Both genotypes demonstrated temporal variations and had significant correlations with rainfall and humidity. CONCLUSION/SIGNIFICANCE: Our study findings suggest that the pathogen specific virulence traits under the influence of ecological factors are the key drivers for geographical variations in the molecular epidemiology of melioidosis.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Melioidose/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidade , Criança , Meio Ambiente , Feminino , Genótipo , Humanos , Índia/epidemiologia , Masculino , Melioidose/epidemiologia , Pessoa de Meia-Idade , Epidemiologia Molecular , Virulência , Adulto Jovem
12.
PLoS Negl Trop Dis ; 13(5): e0007354, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31067234

RESUMO

BACKGROUND: Toll-like receptors (TLRs) are sentinel receptors of the innate immune system. TLR4 detects bacterial lipopolysaccharide (LPS) and TLR5 detects bacterial flagellin. A common human nonsense polymorphism, TLR5:c.1174C>T, results in a non-functional TLR5 protein. Individuals carrying this variant have decreased mortality from melioidosis, infection caused by the flagellated Gram-negative bacterium Burkholderia pseudomallei. Although impaired flagellin-dependent signaling in carriers of TLR5:c.1174C>T is well established, this study tested the hypothesis that a functional effect of TLR5:c.1174C>T is flagellin-independent and involves LPS-TLR4 pathways. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood from two independent cohorts of individuals genotyped at TLR5:c.1174C>T was stimulated with wild type or aflagellated B. pseudomallei or purified bacterial motifs followed by plasma cytokine measurements. Blood from individuals carrying the TLR5:c.1174C>T variant produced less IL-6 and IL-10 in response to an aflagellated B. pseudomallei mutant and less IL-8 in response to purified B. pseudomallei LPS than blood from individuals without the variant. TLR5 expression in THP1 cells was silenced using siRNA; these cells were stimulated with LPS before cytokine levels in cell supernatants were quantified by ELISA. In these cells following LPS stimulation, silencing of TLR5 with siRNA reduced both TNF-α and IL-8 levels. These effects were not explained by differences in TLR4 mRNA expression or NF-κB or IRF activation. CONCLUSIONS/SIGNIFICANCE: The effects of the common nonsense TLR5:c.1174C>T polymorphism on the host inflammatory response to B. pseudomallei may not be restricted to flagellin-driven pathways. Moreover, TLR5 may modulate TLR4-dependent cytokine production. While these results may have broader implications for the role of TLR5 in the innate immune response in melioidosis and other conditions, further studies of the mechanisms underlying these observations are required.


Assuntos
Burkholderia pseudomallei/imunologia , Flagelina/imunologia , Melioidose/genética , Melioidose/imunologia , Polimorfismo Genético , Receptor 5 Toll-Like/genética , Adolescente , Adulto , Idoso , Burkholderia pseudomallei/genética , Códon sem Sentido , Estudos de Coortes , Feminino , Flagelina/genética , Humanos , Imunidade Inata , Interleucina-10/genética , Interleucina-10/imunologia , Masculino , Melioidose/microbiologia , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/imunologia , Mutação Puntual , Receptor 5 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Adulto Jovem
13.
PLoS Negl Trop Dis ; 13(4): e0007348, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002718

RESUMO

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.


Assuntos
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Variação Genética , Melioidose/microbiologia , Monitoramento Ambiental , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Oryza/microbiologia , Prevalência , Microbiologia do Solo , Tailândia , Microbiologia da Água
14.
Medicine (Baltimore) ; 98(9): e14461, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30817562

RESUMO

Burkholderia pseudomallei is the causative agent of meliodosis, and the cases in China are gradually increasing. The present retrospective study aimed to surveil the molecular epidemiological characteristics and antibiotic resistance of B pseudomallei isolates. B pseudomallei strains were isolated and verified from meliodosis patients with relevant epidemiological information from 2004 to 2016 in Hainan, China. Pulsed-field gel electrophoresis based on Spe I digestion was carried out, and antimicrobial resistance of B pseudomallei strains was observed against 9 frequently-used antimicrobials. A total of 164 B pseudomallei isolates were successfully divided into 60 pulsed-field gel electrophoresis (PFGE) patterns, including 33 clusters and 27 single types, at an 85% similarity level. The isolates also exhibited a high level of ceftazidime resistance rate (12.8%, 21/164). B pseudomallei strains were mainly heterogenous with no predominant type, but there were some clonal populations, dominate clusters prevalent and the resistance rates of cephems antimicrobial increased significantly between 2004 and 2016 along with the number of melioidosis cases collected in Hainan (cefoperazone-sulbactam [SCF], rs = 0.96, P = .04; ceftazidime [CAZ], rs = 0.98, P = .01). In conclusion, this study will help to enhance our understanding of molecular characteristics and antibiotic resistance of B pseudomallei.


Assuntos
Burkholderia pseudomallei/genética , Farmacorresistência Bacteriana , Melioidose/epidemiologia , Antibacterianos/farmacologia , Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/isolamento & purificação , Cefoperazona/farmacologia , Ceftazidima/farmacologia , China/epidemiologia , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Masculino , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Prevalência , Estudos Retrospectivos , Sulbactam/farmacologia
15.
Mol Biol (Mosk) ; 53(1): 142-153, 2019.
Artigo em Russo | MEDLINE | ID: mdl-30895962

RESUMO

Evolution of microsatellites (or simple sequence repeats, SSRs) is a complex process that converts perfect repeats to novel structural elements with functions poorly understood, such as imperfect and compound microsatellites. An in silico analysis often Burkholderia pseudomallei genomes revealed 215683 micro-satellites, and more than 98% of them proved imperfect. The density of microsatellites in the genome ranged from 2922.7 to 3022.6 per Mbp. Approximately 10.20-10.67% of the repeats were parts of compound micro-satellites. The of compound microsatellite density varied from 144.7 to 150.6 per Mbp. Between-strain differences in microsatellite distribution were explained by a direct correlation of the SSR density with the GC content and an inverse relationship between the SSR density and the genome size. For each B. pseudomallei chromosome, the SSR density similarly correlated with its size and GC content. Chromosome 2 showed a significant correlation between the SSR and compound microsatellite densities (r = 0.93, p < 10^(-3)). The association of imperfect and compound microsatellite densities with the structural features of each chromosome and the fact that motifs are degenerate and occur in few copies in the majority of B. pseudomallei microsatellites agree with the previous hypothesis of negative selection affecting extended SSRs. The mechanism of selection possibly involves an accumulation of point mutations, which lead to an interruption of the repeat during replication because easily passable secondary structures may form to stabilize the microsatellite length.


Assuntos
Burkholderia pseudomallei/genética , Genoma Bacteriano , Repetições de Microssatélites
16.
Am J Trop Med Hyg ; 100(5): 1082-1084, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30793685

RESUMO

Melioidosis is a tropical infection, first described in Myanmar but now rarely diagnosed there, which is widespread in Southeast Asia. The infection is predominantly acquired by people and animals through contact with soil or water. This study aimed to detect the causative organism, Burkholderia pseudomallei, in environmental samples from farms in Thanlyin and Hmawbi townships near Yangon, Myanmar. One hundred and twenty soil samples and 12 water samples were collected and processed using standard microbiological methods. Burkholderia species were isolated from 50 of the 120 (42%) soil samples but none of the water samples. Arabinose assimilation was tested to differentiate between B. pseudomallei and the nonpathogenic Burkholderia thailandensis, and seven of 50 isolates (14%) were negative. These were all confirmed as B. pseudomallei by a species-specific multiplex polymerase chain reaction (PCR). This is the first study to detect environmental B. pseudomallei in Myanmar and confirms that melioidosis is still endemic in the Yangon area.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Fazendas , Microbiologia do Solo , Arabinose/metabolismo , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/genética , Doenças Endêmicas , Melioidose/epidemiologia , Mianmar , Microbiologia da Água
17.
J Med Microbiol ; 68(2): 263-278, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30628877

RESUMO

PURPOSE: Burkholderia pseudomallei, the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation. METHODOLOGY: Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics.Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R. CONCLUSION: Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei.


Assuntos
Bacteriófagos/genética , Burkholderia pseudomallei/genética , Melioidose/microbiologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/virologia , Clonagem Molecular , Biologia Computacional , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Viral/análise , DNA Viral/química , DNA Viral/isolamento & purificação , Duplicação Gênica/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Humanos , Microscopia Eletrônica , Família Multigênica , Myoviridae/genética , Myoviridae/isolamento & purificação , Myoviridae/ultraestrutura , Fenótipo , RNA Bacteriano/análise , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , Análise de Sequência de DNA , Análise de Sequência de RNA
18.
Int J Antimicrob Agents ; 53(5): 582-588, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30639528

RESUMO

Ceftazidime (CAZ) is the antibiotic of choice for the treatment of Burkholderia pseudomallei infection (melioidosis). The chromosomally-encoded PenA ß-lactamase possesses weak cephalosporinase activity. The wild-type penA gene confers clinically significant CAZ resistance only when overexpressed due to a promoter mutation, transcriptional antitermination or by gene duplication and amplification (GDA). Here we characterise a reversible 33-kb GDA event involving wild-type penA in a CAZ-resistant B. pseudomallei clinical isolate from Thailand. We show that duplication arises from exchanges between short (<10 bp) chromosomal sequences, which in this example consist of 4-bp repeats flanked by 3-bp inverted repeats. GDA involving ß-lactamases may be a common CAZ resistance mechanism in B. pseudomallei.


Assuntos
Antibacterianos/farmacologia , Burkholderia pseudomallei/efeitos dos fármacos , Ceftazidima/farmacologia , Farmacorresistência Bacteriana , Amplificação de Genes , Duplicação Gênica , beta-Lactamases/genética , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/genética , DNA Bacteriano/genética , Humanos , Melioidose/microbiologia , Tailândia
19.
Emerg Infect Dis ; 24(12): 2331-2333, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30457548

RESUMO

We isolated Burkholderia pseudomallei, the causative agent of melioidosis, from liver granulomas of a pet green iguana (Iguana iguana) in Belgium. This case highlights a risk for imported green iguanas acting as a reservoir for introduction of this high-threat, zoonotic pathogen into nonendemic regions.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Iguanas/microbiologia , Melioidose/microbiologia , Animais , Bélgica , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/genética , Feminino , Granuloma/microbiologia , Granuloma/patologia , Fígado/microbiologia , Fígado/patologia , Melioidose/transmissão
20.
PLoS Negl Trop Dis ; 12(11): e0006915, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30418974

RESUMO

BACKGROUND: The Gram-negative soil dwelling bacterium Burkholderia pseudomallei is the etiological agent of melioidosis. The disease is endemic in most parts of Southeast Asia and northern Australia. Over last few years, there has been an increase in number of melioidosis cases from India; however the disease epidemiology is less clearly understood. Multi-locus sequence typing (MLST) is a powerful genotypic method used to characterize the genetic diversity of B. Pseudomallei both within and across the geographic regions. METHODS: In this study, MLST analysis was performed on 64 B. pseudomallei clinical isolates. These isolates were obtained between 2008-2014 from southwestern coastal region of India. Broad population patterns of Indian B. pseudomallei isolates in context with isolates of Southeast Asia or global collection was determined using in silico phylogenetic tools. RESULTS: A total of 32 Sequence types (STs) were reported among these isolates of which 17 STs (53%) were found to be novel. ST1368 was found as group founder and the most predominant genotype (n = 11, 17%). Most of the B. pseudomallei isolates reported in this study (or other Indian isolates available in MLST database) clustered in one major group suggesting clonality in Indian isolates; however, there were a few outliers. When analyzed by measure of genetic differentiation (FST) and other phylogenetic tools (e.g. PHYLOViZ), Indian STs were found closer to Southeast Asian isolates than Australian isolates. The phylogenetic analysis further revealed that within Asian clade, Indian isolates grouped more closely with isolates from Sri Lanka, Vietnam, Bangladesh and Thailand. CONCLUSIONS: Overall, the results of this study suggest that the Indian B. pseudomallei isolates are closely related with lesser heterogeneity among them and cluster in one major group suggesting clonality of the isolates. However, it appears that there are a few outliers which are distantly related to the majority of Indian STs. Phylogenetic analysis suggest that Indian isolates are closely related to isolates from Southeast Asia, particularly from South Asia.


Assuntos
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Genótipo , Melioidose/epidemiologia , Filogenia , Ásia Sudeste/epidemiologia , Austrália/epidemiologia , Burkholderia pseudomallei/classificação , Análise por Conglomerados , DNA Bacteriano/genética , Doenças Endêmicas/prevenção & controle , Doenças Endêmicas/estatística & dados numéricos , Variação Genética , Geografia , Humanos , Índia/epidemiologia , Melioidose/microbiologia , Tipagem de Sequências Multilocus/métodos , Análise de Sequência de DNA
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