Overexpression of the Endothelial Protein C Receptor Is Detrimental during Pneumonia-Derived Gram-negative Sepsis (Melioidosis)

Background

The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia.

Methodology/Principal Findings

Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR^−/−). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR^−/− mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma.

Conclusion/Significance

Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.     

Osteopontin Impairs Host Defense during Established Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)

Background

Melioidosis, caused by infection with Burkholderia (B.) pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN) is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of inflammatory cells.

Methodology and Principal Findings

OPN levels were determined in plasma from 33 melioidosis patients and 31 healthy controls, and in wild-type (WT) mice intranasally infected with B. pseudomallei. OPN function was studied in experimental murine melioidosis using WT and OPN knockout (KO) mice. Plasma OPN levels were elevated in patients with severe melioidosis, even more so in patients who went on to die. In patients who recovered plasma OPN concentrations had decreased after treatment. In experimental melioidosis in mice plasma and pulmonary OPN levels were also increased. Whereas WT and OPN KO mice were indistinguishable during the first 24 hours after infection, after 72 hours OPN KO mice demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, especially due to less necrosis, and decreased neutrophil infiltration. Moreover, OPN KO mice displayed a delayed mortality as compared to WT mice. OPN deficiency did not influence the induction of proinflammatory cytokines.

Conclusions

These data suggest that sustained production of OPN impairs host defense during established septic melioidosis.     

Glyburide Reduces Bacterial Dissemination in a Mouse Model of Melioidosis

Background

Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ∼40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis.

Methods

Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ∼6×10² cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime (600 mg/kg intraperitoneally twice daily starting 24 h after inoculation) in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1β by bone-marrow-derived macrophages (BMDM).

Results

Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1β production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1β by BMDMs in a dose-dependent fashion.

Conclusions

Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1β secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium.     

Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.     

Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei

Background

Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling.

Methods/Principal Findings

An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and ‘low-tech’ methodology that is applicable in both developed and developing countries.

Conclusions/Significance

The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.     

Complete killing of Caenorhabditis elegans by Burkholderia pseudomallei is dependent on prolonged direct association with the viable pathogen

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis.

Methodology/Principal Findings

In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.

Conclusions/Significance

A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.     

MyD88 dependent signaling contributes to protective host defense against Burkholderia pseudomallei

Background

Toll-like receptors (TLRs) have a central role in the recognition of pathogens and the initiation of the innate immune response. Myeloid differentiation primary-response gene 88 (MyD88) and TIR-domain-containing adaptor protein inducing IFNβ (TRIF) are regarded as the key signaling adaptor proteins for TLRs. Melioidosis, which is endemic in SE-Asia, is a severe infection caused by the gram-negative bacterium Burkholderia pseudomallei. We here aimed to characterize the role of MyD88 and TRIF in host defense against melioidosis.

Methodology and Principal Findings

First, we found that MyD88, but not TRIF, deficient whole blood leukocytes released less TNFα upon stimulation with B. pseudomallei compared to wild-type (WT) cells. Thereafter we inoculated MyD88 knock-out (KO), TRIF mutant and WT mice intranasally with B. pseudomallei and found that MyD88 KO, but not TRIF mutant mice demonstrated a strongly accelerated lethality, which was accompanied by significantly increased bacterial loads in lungs, liver and blood, and grossly enhanced liver damage compared to WT mice. The decreased bacterial clearance capacity of MyD88 KO mice was accompanied by a markedly reduced early pulmonary neutrophil recruitment and a diminished activation of neutrophils after infection with B. pseudomallei. MyD88 KO leukocytes displayed an unaltered capacity to phagocytose and kill B. pseudomallei in vitro.

Conclusions

MyD88 dependent signaling, but not TRIF dependent signaling, contributes to a protective host response against B. pseudomallei at least in part by causing early neutrophil recruitment towards the primary site of infection.     

The Concentrations of Ambient Burkholderia Pseudomallei during Typhoon Season in Endemic Area of Melioidosis in Taiwan

Background

Melioidosis is a severe bacterial infection caused by Burkholderia pseudomallei with a high case-fatality rate. Epidemiological and animal studies show the possibility of inhalation transmission. However, no B. pseudomallei concentrations in ambient air have been researched. Here, we developed a method to quantify ambient B. pseudomallei and then measured concentrations of ambient B. pseudomallei during the typhoon season and the non-typhoon season to determine the factors influencing ambient B. pseudomallei levels.

Methods

We quantified ambient B. pseudomallei by using a filter/real-time qPCR method in the Zoynan Region in Kaohsiung, southern Taiwan. Twenty-four hour samples were collected at a sampling rate of 20 L/min every day from June 11 to December 21, 2012 including during the typhoon season (June to September) and reference season (October to December).

Results

We successfully developed a filtration/real-time qPCR method to quantify ambient B. pseudomallei. To our knowledge, this is the first report describing concentrations of ambient B. pseudomallei. Ambient B. pseudomallei were only detected during the typhoon season when compared to the reference season. For the typhoons affecting the Zoynan Region, the positive rates of ambient B. pseudomallei were very high at 80% to 100%. During June to December, rainfall was positively correlated with ambient B. pseudomallei with a statistical significance. Sediment at a nearby pond significantly influenced the concentration of ambient B. pseudomallei. During the typhoon month, the typhoon was positively correlated with ambient B. pseudomallei whereas wind speed was reversely correlated with ambient B. pseudomallei.

Conclusions

Our data suggest the possibility of transmission of B. pseudomallei via inhalation during the typhoon season.     

Burkholderia pseudomallei Is Genetically Diverse in Agricultural Land in Northeast Thailand

Background

The soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined.

Methods

Fixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10×10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).

Principal Findings

B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST). Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations.

Conclusions

Genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.     

Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

Background

The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is the cause of melioidosis, a severe disease of humans and animals in southeast Asia and northern Australia. Despite the detection of B. pseudomallei in various soil and water samples from endemic areas, the environmental habitat of B. pseudomallei remains unclear.

Methodology/Principal Findings

We performed a large survey in the Darwin area in tropical Australia and screened 809 soil samples for the presence of these bacteria. B. pseudomallei were detected by using a recently developed and validated protocol involving soil DNA extraction and real-time PCR targeting the B. pseudomallei–specific Type III Secretion System TTS1 gene cluster. Statistical analyses such as multivariable cluster logistic regression and principal component analysis were performed to assess the association of B. pseudomallei with environmental factors. The combination of factors describing the habitat of B. pseudomallei differed between undisturbed sites and environmentally manipulated areas. At undisturbed sites, the occurrence of B. pseudomallei was found to be significantly associated with areas rich in grasses, whereas at environmentally disturbed sites, B. pseudomallei was associated with the presence of livestock animals, lower soil pH and different combinations of soil texture and colour.

Conclusions/Significance

This study contributes to the elucidation of environmental factors influencing the occurrence of B. pseudomallei and raises concerns that B. pseudomallei may spread due to changes in land use.     

Induction of Mouse Melioidosis with Meningitis by CD11b+ Phagocytic Cells Harboring Intracellular B. pseudomallei as a Trojan Horse

Background

Approximately 3–5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited.

Methods and Findings

We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b⁺ populations. The CD11b⁺Ly6C^high inflamed monocytes, CD11b⁺Ly6C^low resident monocytes, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺F4/80⁺ macrophages and CD11b⁺CD19⁺ B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b⁺ cells induced bacterial colonization in the brain, whereas CD11b⁻ cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b⁺ cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b⁺ cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b⁺ CD62L-negative cells did not.

Conclusions/Significance

We suggest that B. pseudomallei-infected CD11b⁺ selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.     

Molecular phylogeny of Burkholderia pseudomallei from a remote region of Papua New Guinea

Background

The island of New Guinea is located midway between the world''s two major melioidosis endemic regions of Australia and Southeast Asia. Previous studies in Papua New Guinea have demonstrated autochthonous melioidosis in Balimo, Western province. In contrast to other regions of endemicity, isolates recovered from both environmental and clinical sources demonstrate narrow genetic diversity over large spatial and temporal scales.

Methodology/Principal Findings

We employed molecular typing techniques to determine the phylogenetic relationships of these isolates to each other and to others worldwide to aid in understanding the origins of the Papua New Guinean isolates. Multi-locus sequence typing of the 39 isolates resolved three unique sequence types. Phylogenetic reconstruction and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Gene cluster analysis however, identified a Yersinia-like fimbrial gene cluster predominantly found among Burkholderia pseudomallei derived from Southeast Asia. Higher resolution VNTR typing and phylogenetic reconstruction of the Balimo isolates resolved 24 genotypes with long branch lengths. These findings are congruent with long term persistence in the region and a high level of environmental stability.

Conclusions/Significance

Given that anthropogenic influence has been hypothesized as a mechanism for the dispersal of B. pseudomallei, these findings correlate with limited movement of the indigenous people in the region. The palaeogeographical and anthropogenic history of Australasia and the results from this study indicate that New Guinea is an important region for the further study of B. pseudomallei origins and dissemination.     

T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients

Background

Melioidosis is an increasingly recognised cause of sepsis and death across South East Asia and Northern Australia, caused by the bacterium Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal disease, and a vaccine targeting at-risk populations is urgently required. A better understanding of the protective immune response in naturally infected patients is essential for vaccine design.

Methods

We conducted a longitudinal clinical and immunological study of 200 patients with melioidosis on admission, 12 weeks (n = 113) and 52 weeks (n = 65) later. Responses to whole killed B. pseudomallei were measured in peripheral blood mononuclear cells (PBMC) by interferon-gamma (IFN-γ) ELIspot assay and flow cytometry and compared to those of control subjects in the region with diabetes (n = 45) and without diabetes (n = 43).

Results

We demonstrated strong CD4+ and CD8+ responses to B. pseudomallei during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for melioidosis patients, and B. pseudomallei-specific cellular responses in fatal cases (mean 98 IFN-γ cells per million PBMC) were significantly lower than those in the survivors (mean 142 IFN-γ cells per million PBMC) in a multivariable logistic regression model (P = 0.01). A J-shaped curve association between circulating neutrophil count and mortality was seen with an optimal count of 4000 to 8000 neutrophils/μl.Melioidosis patients with known diabetes had poor diabetic control (median glycated haemoglobin HbA_1c 10.2%, interquartile range 9.2–13.1) and showed a stunted B. pseudomallei-specific cellular response during acute illness compared to those without diabetes.

Conclusions

The results demonstrate the role of both CD4+ and CD8+ T-cells in protection against melioidosis, and an interaction between diabetes and cellular responses. This supports development of vaccine strategies that induce strong T-cell responses for the control of intracellular pathogens such as B. pseudomallei.     

Genetic diversity and microevolution of Burkholderia pseudomallei in the environment

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.     

Toll-Like Receptor 4 Stimulation before or after Streptococcus pneumoniae Induced Sepsis Improves Survival and Is Dependent on T-Cells

Introduction

Endotoxin tolerance improves outcomes from gram negative sepsis but the underlying mechanism is not known. We determined if endotoxin tolerance before or after pneumococcal sepsis improved survival and the role of lymphocytes in this protection.

Methods

Mice received lipopolysaccharide (LPS) or vehicle before or after a lethal dose of Streptococcus pneumoniae. Survival, quantitative bacteriology, liver function, and cytokine concentrations were measured. We confirmed the necessity of Toll-like receptor 4 (TLR4) for endotoxin tolerance using C3H/HeN (TLR4 replete) and C3H/HeJ (TLR4 deficient) mice. The role of complement was investigated through A/J mice deficient in C5 complement. CBA/CaHN-Btk^xid//J mice with dysfunctional B cells and Rag-1 knockout (KO) mice deficient in T and B cells delineated the role of lymphocytes.

Results

Endotoxin tolerance improved survival from pneumococcal sepsis in mice with TLR4 that received LPS pretreatment or posttreatment. Survival was associated with reduced bacterial burden and serum cytokine concentrations. Death was associated with abnormal liver function and blood glucose concentrations. Endotoxin tolerance improved survival in A/J and CBA/CaHN-Btk^xid//J mice but not Rag-1 KO mice.

Conclusions

TLR4 stimulation before or after S. pneumoniae infection improved survival and was dependent on T-cells but did not require an intact complement cascade or functional B cells.     

Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance

Background

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear.

Methodology/Principal Findings

The change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition.

Conclusions/Significance

The possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.     

Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

Background

Burkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei.

Methodology and Principal Findings

Using a murine lung epithelial cell line, primary lung epithelial cells and an inhalational murine infection model, we characterized the types of innate immunity proteins and peptides produced upon B. pseudomallei infection. Among a wide panel of immune components studied, increased levels of major pro-inflammatory cytokines IL-6 and TNFα, chemokine MCP-1, and up-regulation of secretory leukocyte protease inhibitor (SLPI) and chemokine (C-C motif) ligand 20 (CCL20) were observed. Inhibition assays using specific inhibitors suggested that NF-κB and p38 MAPK pathways were responsible for these B. pseudomallei-induced antimicrobial peptides.

Conclusions

Our findings indicate that the respiratory epithelial cells, which form the majority of the cells lining the epithelial tract and the lung, have important roles in the innate immune response against B. pseudomallei infection.     

MIF participates in Toxoplasma gondii-induced pathology following oral infection

Background

Macrophage migration inhibitory factor (MIF) is essential for controlling parasite burden and survival in a model of systemic Toxoplasma gondii infection. Peroral T. gondii infection induces small intestine necrosis and death in susceptible hosts, and in many aspects resembles inflammatory bowel disease (IBD). Considering the critical role of MIF in the pathogenesis of IBD, we hypothesized that MIF participates in the inflammatory response induced by oral infection with T. gondii.

Methodology/Principal Findings

Mif deficient (Mif⁻/⁻) and wild-type mice in the C57Bl/6 background were orally infected with T. gondii strain ME49. Mif⁻/⁻ mice had reduced lethality, ileal inflammation and tissue damage despite of an increased intestinal parasite load compared to wt mice. Lack of MIF caused a reduction of TNF-α, IL-12, IFN-γ and IL-23 and an increased expression of IL-22 in ileal mucosa. Moreover, suppressed pro-inflammatory responses at the ileal mucosa observed in Mif⁻/⁻ mice was not due to upregulation of IL-4, IL-10 or TGF-β. MIF also affected the expression of matrix metalloproteinase-9 (MMP-9) but not MMP-2 in the intestine of infected mice. Signs of systemic inflammation including the increased concentrations of inflammatory cytokines in the plasma and liver damage were less pronounced in Mif⁻/⁻ mice compared to wild-type mice.

Conclusion/Significance

In conclusion, our data suggested that in susceptible hosts MIF controls T. gondii infection with the cost of increasing local and systemic inflammation, tissue damage and death.