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Burkholderia pseudomallei Is Spatially Distributed in Soil in Northeast Thailand  [PDF]
Direk Limmathurotsakul ,Vanaporn Wuthiekanun,Narisara Chantratita,Gumphol Wongsuvan,Premjit Amornchai,Nicholas P. J. Day,Sharon J. Peacock
PLOS Neglected Tropical Diseases , 2010, DOI: 10.1371/journal.pntd.0000694
Abstract: Background Melioidosis is a frequently fatal infectious disease caused by the soil dwelling Gram-negative bacterium Burkholderia pseudomallei. Environmental sampling is important to identify geographical distribution of the organism and related risk of infection to humans and livestock. The aim of this study was to evaluate spatial distribution of B. pseudomallei in soil and consider the implications of this for soil sampling strategies. Methods and Findings A fixed-interval sampling strategy was used as the basis for detection and quantitation by culture of B. pseudomallei in soil in two environmental sites (disused land covered with low-lying scrub and rice field) in northeast Thailand. Semivariogram and indicator semivariogram were used to evaluate the distribution of B. pseudomallei and its relationship with range between sampling points. B. pseudomallei was present on culture of 80/100 sampling points taken from the disused land and 28/100 sampling points from the rice field. The median B. pseudomallei cfu/gram from positive sampling points was 378 and 700 for the disused land and the rice field, respectively (p = 0.17). Spatial autocorrelation of B. pseudomallei was present, in that samples taken from areas adjacent to sampling points that were culture positive (negative) for B. pseudomallei were also likely to be culture positive (negative), and samples taken from areas adjacent to sampling points with a high (low) B. pseudomallei count were also likely to yield a high (low) count. Ranges of spatial autocorrelation in quantitative B. pseudomallei count were 11.4 meters in the disused land and 7.6 meters in the rice field. Conclusions We discuss the implications of the uneven distribution of B. pseudomallei in soil for future environmental studies, and describe a range of established geostatistical sampling approaches that would be suitable for the study of B. pseudomallei that take account of our findings.
Activation of Toll-like receptors by Burkholderia pseudomallei
T Eoin West, Robert K Ernst, Malinka J Jansson-Hutson, Shawn J Skerrett
BMC Immunology , 2008, DOI: 10.1186/1471-2172-9-46
Abstract: In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6) and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-α and MIP-2 production whereas in TLR4-/- cells, TNF-α, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-α production in response to heat-killed B. pseudomallei.B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88-dependent signaling pathways may also be important in the host response to B. pseudomallei infection. These findings provide new insights into critical mechanisms of host defense in melioidosis.Melioidosis is an endemic and poorly understood infectious disease in much of the tropical world; it is particularly prevalent in east Asia and northern Australia. The disease accounts for 20% of community-acquired sepsis in parts of northeast Thailand. Despite antibiotic treatment, mortality rates approach 40% [1]. The causative organism, Burkholderia pseudomallei (Bp), is a Gram-negative environmental saprophyte. Aerosol or transcutaneous infection results in an extensive range of disease – from chronic, relapsing illness with abscess formation to fulminant pneumonia and septicemia [2]. The lung is the most commonly affected organ. Concern about the use of Bp as a bioweapon has led to its classification as a CDC Category B pathogen. While t
Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment  [PDF]
Narisara Chantratita equal contributor,Vanaporn Wuthiekanun equal contributor,Direk Limmathurotsakul,Mongkol Vesaratchavest,Aunchalee Thanwisai,Premjit Amornchai,Sarinna Tumapa,Edward J. Feil,Nicholas P. Day,Sharon J. Peacock
PLOS Neglected Tropical Diseases , 2008, DOI: 10.1371/journal.pntd.0000182
Abstract: Background The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined. Methods and Findings We undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m2 of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Twelve PFGE types and nine sequence types (STs) were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively), only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93). Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone. Conclusions We discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.
Global transcriptional profiling of Burkholderia pseudomallei under salt stress reveals differential effects on the Bsa type III secretion system
Pornpan Pumirat, Jon Cuccui, Richard A Stabler, Joanne M Stevens, Veerachat Muangsombut, Ekapot Singsuksawat, Mark P Stevens, Brendan W Wren, Sunee Korbsrisate
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-171
Abstract: Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system.B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei.Burkholderia pseudomallei is a saprophyte and the causative agent of melioidosis, a human infectious disease endemic in some tropical areas including southeast Asia and northern Australia [1]. Inhalation is a recognized route of infection with this organism and pulmonary disease is common [1,2]. Owing to its aerosol infectivity, the severe course of infection, and the absence of vaccines and fully effective treatments, B. pseudomallei is classified as a hazard category three pathogen and considered a potential biothreat agent [2]. B. pseudomallei, is a Gram negative bacillus found in soil and water over a wide endemic area and mainly infects people who have direct contact with wet soil [1,3]. In Thailand, the highest incidence of melioidosis is in the northeast region, at a rate of approximately 3.6-5.5 per 100,000 human populations annually. Septicaemic presentati
Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors  [PDF]
Andrew P. Liguori, Stephanie D. Warrington, Jennifer L. Ginther, Talima Pearson, Jolene Bowers, Mindy B. Glass, Mark Mayo, Vanaporn Wuthiekanun, David Engelthaler, Sharon J. Peacock, Bart J. Currie, David M. Wagner, Paul Keim, Apichai Tuanyok
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0029323
Abstract: Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.
Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei  [PDF]
Anuwat Aunkham, Albert Schulte, Mathias Winterhalter, Wipa Suginta
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0095918
Abstract: Background Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins. Principal Findings Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive. Conclusion/Significance Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.
Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro
Sarunporn Tandhavanant, Aunchalee Thanwisai, Direk Limmathurotsakul, Sunee Korbsrisate, Nicholas PJ Day, Sharon J Peacock, Narisara Chantratita
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-303
Abstract: Morphotype was associated with survival in the presence of H2O2 and antimicrobial peptide LL-37, but not with susceptibility to acid, acidified sodium nitrite, or resistance to lysozyme, lactoferrin, human neutrophil peptide-1 or human beta defensin-2. Incubation under anaerobic conditions was a strong driver for switching of type III to an alternative morphotype. Differences were noted in the survival and replication of the three types following uptake by human macrophages, but marked strain-to strain-variability was observed. Uptake of type III alone was associated with colony morphology switching.Morphotype is associated with phenotypes that alter the ability of B. pseudomallei to survive in adverse environmental conditions.Burkholderia pseudomallei is an environmental Gram-negative bacterium that causes a severe and often fatal disease called melioidosis. This is an important cause of sepsis in south-east Asia and northern Australia, a geographic distribution that mirrors the presence of B. pseudomallei in the environment [1]. Melioidosis may develop following bacterial inoculation or inhalation and occurs most often in people with regular contact with contaminated soil and water [1]. Clinical manifestations of melioidosis are highly variable and range from fulminant septicemia to mild localized infection. The overall mortality rate is 40% in northeast Thailand (rising to 90% in patients with severe sepsis) and 20% in northern Australia [1,2].A major feature of melioidosis is that bacterial eradication is difficult to achieve. Fever clearance time is often prolonged (median 8 days), antimicrobial therapy is required for 12-20 weeks, and relapse occurs in around 10% of patients despite an appropriate course of antimicrobial therapy [3,4]. The basis for persistence in the infected human host is unknown, although several observations made to date may be relevant to the clinical behaviour of this organism [2,5]. B. pseudomallei can resist the action of bactericidal
Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia pseudomallei Clinical Isolates from Thailand  [PDF]
Lily A. Trunck,Katie L. Propst,Vanaporn Wuthiekanun,Apichai Tuanyok,Stephen M. Beckstrom-Sternberg,James S. Beckstrom-Sternberg,Sharon J. Peacock,Paul Keim,Steven W. Dow,Herbert P. Schweizer
PLOS Neglected Tropical Diseases , 2009, DOI: 10.1371/journal.pntd.0000519
Abstract: Background Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a. Methodology/Principal Findings qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s). Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively). This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model. Conclusions/Significance Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Δ(amrAB-oprA) mutants in such experiments.
Evolution of Burkholderia pseudomallei in Recurrent Melioidosis  [PDF]
Hillary S. Hayden, Regina Lim, Mitchell J. Brittnacher, Elizabeth H. Sims, Elizabeth R. Ramage, Christine Fong, Zaining Wu, Eva Crist, Jean Chang, Yang Zhou, Matthew Radey, Laurence Rohmer, Eric Haugen, Will Gillett, Vanaporn Wuthiekanun, Sharon J. Peacock, Rajinder Kaul, Samuel I. Miller, Colin Manoil, Michael A. Jacobs
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036507
Abstract: Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.
Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection  [cached]
Sarovich DS,Price EP,Limmathurotsakul D,Cook JM
Infection and Drug Resistance , 2012,
Abstract: Derek S Sarovich,1,2,* Erin P Price,1,2,* Direk Limmathurotsakul,3 James M Cook,1 Alex T Von Schulze,1 Spenser R Wolken,1 Paul Keim,1 Sharon J Peacock,3,4 Talima Pearson1 1Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA; 2Tropical and Emerging Infectious Diseases Division, Menzies School of Health Research, Darwin, Australia; 3Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 4Department of Medicine, University of Cambridge, Cambridge, United Kingdom*These authors contributed equally to this workAbstract: Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease.Keywords: Burkholderia pseudomallei, ceftazidime, antibiotic resistance, melioidosis, β-lactamase, penA
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