Burkholderia pseudomallei Is Genetically Diverse in Agricultural Land in Northeast Thailand

Burkholderia pseudomallei is the cause of melioidosis, a serious human infection most commonly diagnosed in southeast Asia and northern Australia. The organism lives in the soil in a specific geographical distribution and infection results from bacterial inoculation, inhalation or ingestion. The purpose of this study was to define the distribution and genetic diversity of B. pseudomallei in agricultural land where most human infections probably occur. We performed soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within a rice paddy in northeast Thailand, and undertook genotyping of primary culture plate colonies from 11 sampling points. We identified 7 different genotypes, with relatively limited overlap between different sampling points. Two samples contained more than one B. pseudomallei genotype, in which a numerically dominant genotype coexisted with one or more additional genotypes present as a minority population. We conclude that genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings inform future efforts to define B. pseudomallei in the environment, and should be considered during the design stage of studies comparing B. pseudomallei isolated from the environment and from patients with invasive disease

TLR4 genetic variation is associated with inflammatory responses in Gram-positive sepsis.

OBJECTIVES: To identify important pathogen recognition receptor (PRR) pathways regulating innate immune responses and outcome in Staphylococcus aureus sepsis. METHODS: We analysed whether candidate PRR pathway genetic variants were associated with killed S. aureus-induced cytokine responses ex vivo and performed follow-up in vitro studies. We tested the association of our top-ranked variant with cytokine responses and clinical outcomes in a prospective multicentre cohort of patients with staphylococcal sepsis. RESULTS: An intronic TLR4 polymorphism and expression quantitative trait locus, rs1927907, was highly associated with cytokine release induced by stimulation of blood from healthy Thai subjects with S. aureus ex vivo. S. aureus did not induce TLR4-dependent NF-κB activation in transfected HEK293 cells. In monocytes, tumor necrosis factor (TNF)-α release induced by S. aureus was not blunted by a TLR4/MD-2 neutralizing antibody, but in a monocyte cell line, TNF-α was reduced by knockdown of TLR4. In Thai patients with staphylococcal sepsis, rs1927907 was associated with higher interleukin (IL)-6 and IL-8 levels as well as with respiratory failure. S. aureus-induced responses in blood were most highly correlated with responses to Gram-negative stimulants whole blood. CONCLUSIONS: A genetic variant in TLR4 is associated with cytokine responses to S. aureus ex vivo and plasma cytokine levels and respiratory failure in staphylococcal sepsis. While S. aureus does not express lipopolysaccharide or activate TLR4 directly, the innate immune response to S. aureus does appear to be modulated by TLR4 and shares significant commonality with that induced by Gram-negative pathogens and lipopolysaccharide

Co-evolutionary Signals Identify Burkholderia pseudomallei Survival Strategies in a Hostile Environment

The soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and subtropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies (2,011 genomes), condition-wide transcriptome analyses (82 diverse conditions), and a gene knockout assay to uncover signals of "co-selection"-that is a combination of genetic markers that have been repeatedly selected together through B. pseudomallei evolution. These enabled us to identify 13,061 mutation pairs under co-selection in distinct genes and noncoding RNA. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions, highlighting the conditions as one of the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed a BPSL1661 role under nutrient deprivation, and explored the functional basis of co-selection gene network surrounding BPSL1661 in facilitating the bacterial survival under nutrient depletion. Our findings suggest that nutrient-limited conditions have been the common selection pressure acting on this species, and allelic variation of BPSL1661 may have promoted B. pseudomallei survival during harsh environmental conditions by facilitating bacterial adherence to different surfaces, cells, or living hosts.Peer reviewe

Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

The soil dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis, a serious human infection that occurs in Southeast Asia and northern Australia. The purpose of this study was to evaluate the population genetic structure of B. pseudomallei in the environment. To achieve this, we undertook soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within an area of disused land in northeast Thailand, and undertook detailed genotyping of primary plate colonies isolated from three independent sampling points. Our results demonstrated that multiple B. pseudomallei genotypes were present within a single soil sample, and that different genotypes were present at independent but nearby sampling points. The B. pseudomallei genetic population was unevenly distributed within a given sample, with a predominant genotype co-existing with several genotypes present as a minority population. We discuss the implications of this structuring of genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies

Defining the True Sensitivity of Culture for the Diagnosis of Melioidosis Using Bayesian Latent Class Models

BACKGROUND: Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests. METHODS/PRINCIPAL FINDINGS: Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard. CONCLUSIONS/SIGNIFICANCE: Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections

A Simple Scoring System to Differentiate between Relapse and Re-Infection in Patients with Recurrent Melioidosis

Melioidosis is a serious infectious disease caused by the Gram-negative bacterium, Burkholderia pseudomallei. This organism is present in the environment in areas where melioidosis is endemic (most notably East Asia and Northern Australia), and infection is acquired following bacterial inoculation or inhalation. Despite prolonged oral eradicative treatment, recurrent melioidosis occurs in approximately 10% of survivors of acute melioidosis. Recurrent melioidosis can be caused by relapse (failure of initial eradicative treatment) or re-infection with a new infection. The aim of this study was to develop a simple scoring system to distinguish between re-infection and relapse, since this has implications for antimicrobial treatment of the recurrent episode, but telling the two apart normally requires bacterial genotyping. A prospective study of melioidosis patients in NE Thailand conducted between 1986 and 2005 identified 141 patients with recurrent melioidosis. Of these, 92 patients had relapse and 49 patients had re-infection as confirmed by genotyping techniques. We found that relapse was associated with previous inadequate treatment and shorter time to clinical features of recurrence, while re-infection was associated with renal insufficiency and presentation during the rainy season. A simple scoring index to help distinguish between relapse and re-infection was developed to provide important bedside information where rapid bacterial genotyping is unavailable. Guidelines are provided on how this scoring system could be implemented

Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)

Willem Wiersinga and colleagues find up-regulation of multiple Toll-like receptors (TLRs) in peripheral blood cells of patients with melioidosis. However, only TLR2 had an effect on the immune response in a mouse model

Interleukin 10 inhibits pro-inflammatory cytokine responses and killing of Burkholderia pseudomallei.

Melioidosis, caused by Burkholderia pseudomallei, is endemic in northeastern Thailand and Northern Australia. Severe septicemic melioidosis is associated with high levels of pro-inflammatory cytokines and is correlated with poor clinical outcomes. IL-10 is an immunoregulatory cytokine, which in other infections can control the expression of pro-inflammatory cytokines, but its role in melioidosis has not been addressed. Here, whole blood of healthy seropositive individuals (n = 75), living in N. E. Thailand was co-cultured with B. pseudomallei and production of IL-10 and IFN-γ detected and the cellular sources identified. CD3- CD14+ monocytes were the main source of IL-10. Neutralization of IL-10 increased IFN-γ, IL-6 and TNF-α production and improved bacteria killing. IFN-γ production and microbicidal activity were impaired in individuals with diabetes mellitus (DM). In contrast, IL-10 production was unimpaired in individuals with DM, resulting in an IL-10 dominant cytokine balance. Neutralization of IL-10 restored the IFN-γ response of individuals with DM to similar levels observed in healthy individuals and improved killing of B. pseudomallei in vitro. These results demonstrate that monocyte derived IL-10 acts to inhibit potentially protective cell mediated immune responses against B. pseudomallei, but may also moderate the pathological effects of excessive cytokine production during sepsis