Melioidosis

BackgroundMelioidosis therapy is divided into an intravenous intensive phase and an oral eradication phase. The Darwin melioidosis treatment guideline has evolved over two decades, with over 1150 consecutive patients with culture-confirmed melioidosis managed under the Darwin Prospective Melioidosis Study. The current guideline, published in 2015, has been associated with low rates of recrudescence, relapse and mortality, and together with the treatment trials in Thailand, forms the basis for consensus global guidelines. We aimed to reassess the Darwin guideline and determine if any adjustments to the recommendations better reflect current practice in melioidosis therapy at Royal Darwin Hospital.Methodology/principal findingsThis retrospective cohort study reviews the characteristics, admission duration, duration of intravenous antibiotics, recrudescence, recurrence and mortality in all patients presenting with first episode culture-confirmed melioidosis in the tropical north of Australia's Northern Territory from 1st October 2012 until 1st January 2017. 234 patients were available for analysis. 16 (6.8%) died during the intensive phase treatment and 6 (2.6%) did not have complete treatment at Royal Darwin Hospital, leaving 212 patients for analysis. Six (2.8%) patients had recrudescence during therapy and 10 (4.7%) had recurrent melioidosis (relapse or new infection) after completion of therapy. Persisting osteomyelitis requiring surgery was an important reason for recrudescence as was unrecognized osteomyelitis for relapse. For patients presenting with an antibiotic duration determining focus of pneumonia, durations of intravenous antibiotics were often prolonged beyond the current 2-week minimum treatment recommendation. Prolongation of therapy in pneumonia mostly occurred in patients presenting with multi-lobar disease or with concurrent blood culture positivity.Conclusions/significanceThe 2015 Darwin melioidosis guideline is working well with low rates of recrudescence, relapse and mortality. Based on the practice of the treating clinicians, the 2020 revision of the guideline has been adjusted to include a duration of a minimum of 3 weeks of intravenous antibiotics for those with concurrent bacteraemia and pneumonia involving only a single lobe and those with bilateral and unilateral multi-lobar pneumonias who do not have bacteraemia. We also extend to a minimum of 4 weeks intravenous therapy for those with concurrent bacteraemia and bilateral or unilateral multi-lobar pneumonia.

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm.

Diabetes mellitus (DM) is a serious global health problem currently affecting over 450 million people worldwide. Defining its interaction with major global infections is an international public health priority. Melioidosis is caused by Burkholderia pseudomallei, an exemplar pathogen for studying intracellular bacterial infection in the context of DM due to the 12-fold increased risk in this group. We characterized immune correlates of survival in peripheral blood of acute melioidosis patients with and without DM and highlight different immune response patterns. We demonstrate the importance of circulating NK cells and show that CX3CR1 expression on lymphocytes is a novel correlate of survival from acute melioidosis. Furthermore, excessive serum levels of IL-15 and IL-18BP contribute to poor outcome independent of DM comorbidity. CD8+ T cells and granzyme B expression in NK cells are important for survival of non-DM patients, whereas high antibody titers against B. pseudomallei and double-negative T cells are linked to survival of DM patients. Recall responses support a role of γδ T-cell-derived IFN-γ in the establishment of protective immunity in the DM group. Defining the hallmarks of protection in people with DM is crucial for the design of new therapies and vaccines targeting this rapidly expanding risk group.

BackgroundBurkholderia pseudomallei is an environmental bacterium that causes melioidosis, a life-threatening disease prevalent in tropical regions. Accurate detection of B. pseudomallei in the environment is essential for identifying areas that pose an infection risk. This study aimed to evaluate culture- and real-time polymerase chain reaction (PCR)-based methods for detecting B. pseudomallei directly in soil samples, and to assess its environmental prevalence in relation to antibody levels in healthy individuals from Amnat Charoen, Northeast Thailand.Methodology/principal findingsInitial studies used spiked soil samples to evaluate culture-based methods including Ashdown agar, phosphate-buffered acidic erythritol (ACER) agar, Threonine-basal salt solution with colistin 50 (TBSS-C50) broth, TBSS-C50 based erythritol broth, and ACER broth, and real-time PCR assays targeting BPSS1187 and TTS1-orf2. Hemolysin coregulated protein 1 (Hcp1)-specific antibodies were measured in 398 plasma samples using an enzyme-linked immunosorbent assay. Based on these antibody titers, 238 soil samples were collected from the households of individuals that were strongly positive (N = 6) and negative (N = 6) for Hcp1-specific antibodies. B. pseudomallei was isolated from 3.36% (8/238) of soil samples by direct culture on Ashdown agar and from 5.46% (13/238) after enrichment in ACER broth followed by culture on Ashdown agar. Real-time PCR assays confirmed the presence of B. pseudomallei in 2 out of 20 culture-positive soil samples. Overall, B. pseudomallei was detected in 14 of 129 samples (10.9%) from households of seropositive individuals and in 6 of 108 samples (5.5%) from households of seronegative individuals (P = 0.165).Conclusions/significanceThis study demonstrates the importance of using effective methods for detection of B. pseudomallei in environmental samples. For culture-based approaches, enrichment in ACER broth followed by subculture on Ashdown agar demonstrated enhanced bacterial recovery compared to direct culture on Ashdown agar alone. There was limited detection of B. pseudomallei in soil by real-time PCR, and lack of an association between environmental detection methods and human seroprevalence. These findings highlight the need for integrated, multi-dimensional surveillance approaches.