Longitudinal profiling of plasma cytokines in melioidosis and their association with mortality: a prospective cohort study.

OBJECTIVES: To characterize plasma cytokine responses in melioidosis and analyse their association with mortality. METHODS: A prospective longitudinal study was conducted in two hospitals in Northeast Thailand to enrol 161 individuals with melioidosis, plus 13 uninfected healthy individuals and 11 uninfected individuals with diabetes to act as controls. Blood was obtained from all individuals at enrolment (day 0), and at days 5, 12 and 28 from surviving melioidosis patients. Interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-23, and tumour necrosis factor-α (TNF-α) were assayed in plasma. The association of each cytokine and its dynamics with 28-day mortality was determined. RESULTS: Of the individuals with melioidosis, 131/161 (81%) were bacteraemic, and 68/161 (42%) died. On enrolment, median levels of IFN-γ, IL-6, IL-8, IL-10, IL-23 and TNF-α were higher in individuals with melioidosis compared with uninfected healthy individuals and all but IFN-γ were positively associated with 28-day mortality. Interleukin-8 provided the best discrimination of mortality (area under the receiver operating characteristic curve 0.78, 95% CI 0.71-0.85). Over time, non-survivors had increasing IL-6, IL-8 and IL-17A levels, in contrast to survivors. In joint modelling, temporal trajectories of IFN-γ, IL-6, IL-8, IL-10 and TNF-α predicted survival. CONCLUSIONS: In a severely ill cohort of individuals with melioidosis, specific pro- and anti-inflammatory and T helper type 17 cytokines were associated with survival from melioidosis, at enrolment and over time. Persistent inflammation preceded death. These findings support further evaluation of these mediators as prognostic biomarkers and to guide targeted immunotherapeutic development for severe melioidosis.

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.

Comparison of community-onset Staphylococcus argenteus and Staphylococcus aureus sepsis in Thailand: a prospective multicentre observational study.

Staphylococcus argenteus is a globally distributed cause of human infection, but diagnostic laboratories misidentify this as Staphylococcus aureus. We determined whether there is clinical utility in distinguishing between the two. A prospective cohort study of community-onset invasive staphylococcal sepsis was conducted in adults at four hospitals in northeast Thailand between 2010 and 2013. Of 311 patients analysed, 58 (19%) were infected with S. argenteus and 253 (81%) with S. aureus. Most S. argenteus (54/58) were multilocus sequence type 2250. Infection with S. argenteus was more common in males, but rates of bacteraemia and drainage procedures were similar in the two groups. S. argenteus precipitated significantly less respiratory failure than S. aureus (5.2% versus 20.2%, adjusted OR 0.21, 95% CI 0.06-0.74, p 0.015), with a similar but non-significant trend for shock (6.9% versus 12.3%, adjusted OR 0.46, 95% CI 0.15-1.44, p 0.18). This did not translate into a difference in death at 28 days (6.9% versus 8.7%, adjusted OR 0.80, 95% CI 0.24-2.65, p 0.72). S. argenteus was more susceptible to antimicrobial drugs compared with S. aureus, and contained fewer toxin genes although pvl was detected in 16% (9/58). We conclude that clinical differences exist in association with sepsis due to S. argenteus versus S. aureus.

Role of Burkholderia pseudomallei biofilm formation and lipopolysaccharide in relapse of melioidosis.

We examined whether quantitative biofilm formation and/or lipopolysaccharide type of Burkholderia pseudomallei was associated with relapsing melioidosis. We devised a 1:4 nested case-control study in which both cases and controls were drawn from a cohort of patients with primary melioidosis. Paired isolates from 80 patients with relapse and single isolates from 184 patients without relapse were tested. Relapse was associated with biofilm formation of the primary infecting isolate (conditional OR 2.03; 95% CI 1.27-3.25; p 0.003), but not with lipopolysaccharide type (p 0.74). This finding highlights the importance of biofilm formation in relapsing melioidosis.

Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis.

Melioidosis is a severe infection caused by the flagellated bacterium Burkholderia pseudomallei. The nonsense polymorphism TLR51174C>T is associated with improved outcome in Thais with melioidosis. We hypothesized that other TLR5 variants may modulate the host response and determine outcome in melioidosis. We genotyped 12 TLR5 variants selected de novo from the HapMap database and examined the association of each with cytokines induced by flagellin stimulation of whole blood from healthy Thai subjects. We found a blunted cytokine response for three related markers that were in linkage disequilibrium (LD) with a non-synonymous variant, TLR51846T>C. Carriers of TLR51846T>C had broadly impaired cytokine responses induced by flagellin. TLR51846T>C was associated with protection against death in melioidosis patients (odds ratio: 0.62, 95% confidence interval: 0.42-0.93, P=0.021). We observed no impairment in TLR51846C-dependent nuclear factor κB activation, however, suggesting an alternative mechanism for the effect. We found that TLR51846T>C was in strong LD with TLR51174C>T. Many of the blunted cytokine responses observed and the association of TLR51846T>C with survival in melioidosis patients may be attributable to TLR51174C>T, but we could not exclude an independent effect of TLR51846T>C. These data identify novel associations for TLR51846T>C, enhance our understanding of TLR5 genetic architecture in Thais and highlight the role of TLR5 in melioidosis.

DISCRIMINATION OF Burkholderia mallei/pseudomallei FROM Burkholderia thailandensis BY SEQUENCE COMPARISON OF A FRAGMENT OF THE RIBOSOMAL PROTEIN S21 (RPSU) GENE.

Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene.The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of 'out-stander' reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently.Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei.

Bacterial gene loss as a mechanism for gain of antimicrobial resistance.

Acquisition of exogenous DNA by pathogenic bacteria represents the basis for much of the acquired antimicrobial resistance in pathogenic bacteria. A more extreme mechanism to avoid the effect of an antibiotic is to delete the drug target, although this would be predicted to be rare since drug targets are often essential genes. Here, we review and discuss the description of a novel mechanism of resistance to the cephalosporin drug ceftazidime caused by loss of a penicillin-binding protein (PBP) in a Gram-negative bacillus (Burkholderia pseudomallei). This organism causes melioidosis across south-east Asia and northern Australia, and is usually treated with two or more weeks of ceftazidime followed by oral antibiotics for three to six months. Comparison of clinical isolates from six patients with melioidosis found initial ceftazidime-susceptible isolates and subsequent ceftazidime-resistant variants. The latter failed to grow on commonly used culture media, rendering these isolates difficult to detect in the diagnostic laboratory. Genomic analysis using pulsed-field gel electrophoresis and array based genomic hybridisation revealed a large-scale genomic deletion comprising 49 genes in the ceftazidime-resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a PBP 3 present within the region of genomic loss. This provides one explanation for ceftazidime treatment failure, and may be a frequent but undetected event in patients with melioidosis.

Toll-like receptor 4 region genetic variants are associated with susceptibility to melioidosis.

Melioidosis is a tropical infection caused by the Gram-negative soil saprophyte Burkholderia pseudomallei. Despite broad exposure of northeastern Thais, disease develops in only a small proportion of individuals. Although diabetes is a risk factor, the mechanisms of host susceptibility to melioidosis are still poorly understood. We postulated that Toll-like receptors (TLRs) regulate host susceptibility to disease, and that genetic variation in TLRs is associated with melioidosis. We analyzed the frequency of eight previously described TLR pathway polymorphisms in 490 cases compared with 950 non-hospitalized controls or 458 hospitalized controls. Based on these results, we then analyzed the frequency of additional TLR4 or TLR6-1-10 region polymorphisms in cases and controls. We found that the TLR4(1196C>T) variant was associated with protection from melioidosis when compared with non-hospitalized controls. The TLR1(742A>G) and TLR1(-7202A>G) variants were associated with melioidosis when compared with hospitalized controls. In further analyses, we found that two additional TLR4 region polymorphisms were associated with disease. In diabetics, three other TLR6-1-10 region polymorphisms were associated with disease when compared with hospitalized controls. We conclude that TLR genetic variants may modulate host susceptibility to melioidosis. Confirmation of these findings and further investigation of the mechanisms are required.