Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

Burkholderia thailandensis is a non-pathogenic environmental saprophyte closely related to Burkholderia pseudomallei, the causative agent of the often fatal animal and human disease melioidosis. To study B. thailandensis genomic variation, we profiled 50 isolates using a pan-genome microarray comprising genomic elements from 28 Burkholderia strains and species.

In their article, Baelani and colleagues surveyed anesthesia providers from African low- and middle-income countries (LMICs) to evaluate whether or not the current Surviving Sepsis Campaign (SSC) guidelines are feasible in such resource-constrained settings. The authors report that an alarmingly low percentage of hospitals have the capacity to implement the SSC guidelines in their entirety but a higher percentage are able to implement the majority of SSC guidelines and grade 1 recommendations. In reality, the probability of adherence to SSC guidelines for septic management is even lower than reported, given that the majority of sepsis management in African LMICs is likely performed by non-intensivists outside of intensive care units. Efforts to address the challenges of managing severely ill patients in LMICs have recently been taken on by the World Health Organization. After reviewing available evidence for sepsis management predominantly from high-income countries, a panel of experts developed a consensus-based strategy tailored for resource-limited settings. However, more research that can evaluate the challenges specific to sepsis management in LMICs and not currently addressed by the SSC guidelines is needed. Comprehensive, evidence-based guidelines combined with innovative approaches to sepsis management in LMICs are required to make a meaningful impact on worldwide sepsis survival.

Rapid PCR-based tests for the diagnosis of leptospirosis can provide information that contributes towards early patient management, but these have not been adopted in Thailand. Here, we compare the diagnostic sensitivity and specificity of two real-time PCR assays targeting rrs or lipL32 for the diagnosis of leptospirosis in northeast Thailand.

Patients with diabetes mellitus are more prone to bacterial sepsis, but there are conflicting data on whether outcomes are worse in diabetics after presentation with sepsis. Glyburide is an oral hypoglycemic agent used to treat diabetes mellitus. This K(ATP)-channel blocker and broad-spectrum ATP-binding cassette (ABC) transporter inhibitor has broad-ranging effects on the immune system, including inhibition of inflammasome assembly and would be predicted to influence the host response to infection.

The gram-negative bacillus Burkholderia pseudomallei is a saprophyte and the cause of melioidosis. Natural infection is most commonly reported in northeast Thailand and northern Australia but also occurs in other parts of Asia, South America, and the Caribbean. Melioidosis develops after bacterial inoculation or inhalation, often in relation to occupational exposure in areas where the disease is endemic. Clinical infection has a peak incidence between the fourth and fifth decades; with diabetes mellitus, excess alcohol consumption, chronic renal failure, and chronic lung disease acting as independent risk factors. Most affected adults ( approximately 80%) in northeast Thailand, northern Australia, and Malaysia have >/=1 underlying diseases. Symptoms of melioidosis may be exhibited many years after exposure, commonly in association with an alteration in immune status. Manifestations of disease are extremely broad ranging and form a spectrum from rapidly life-threatening sepsis to chronic low-grade infection. A common clinical picture is that of sepsis associated with bacterial dissemination to distant sites, frequently causing concomitant pneumonia and liver and splenic abscesses. Infection may also occur in bone, joints, skin, soft tissue, or the prostate. The clinical symptoms of melioidosis mimic those of many other diseases; thus, differentiating between melioidosis and other acute and chronic bacterial infections, including tuberculosis, is often impossible. Confirmation of the diagnosis relies on good practices for specimen collection, laboratory culture, and isolation of B. pseudomallei. The overall mortality rate of infected persons is 50% in northeast Thailand (35% in children) and 19% in Australia.

Burkholderia pseudomallei, an environmental gram-negative bacillus, is the causative agent of melioidosis and a bio-threat agent. Reports of B. pseudomallei isolation from soil and animals in East and West Africa suggest that melioidosis might be more widely distributed than previously thought. Because it has been found in equatorial areas with tropical climates, we hypothesized that B. pseudomallei could exist in Gabon. During 2012-2013, we conducted a seroprevalance study in which we set up microbiology facilities at a large clinical referral center and prospectively screened all febrile patients by conducting blood cultures and testing for B. pseudomallei and related species; we also determined whether B. pseudomallei could be isolated from soil. We discovered a novel B. pseudomallei sequence type that caused lethal septic shock and identified B. pseudomallei and B. thailandensis in the environment. Our data suggest that melioidosis is emerging in Central Africa but is unrecognized because of the lack of diagnostic microbiology facilities.

Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results.

The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.

The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy.

Neutrophils release neutrophil extracellular traps (NETs) in response to invading pathogens. Although NETs play an important role in host defense against microbial pathogens, they have also been shown to play a contributing mechanistic role in pathologic inflammation in the absence of infection. Although a role for NETs in bacterial pneumonia and acute respiratory distress syndrome (ARDS) is emerging, a comprehensive evaluation of NETs in the alveolar space of critically ill patients has yet to be reported. In this study, we evaluated whether markers of NET formation in mechanically ventilated patients are associated with ventilator-associated pneumonia (VAP).

Limited data are available on sepsis in low-resource settings, particularly outside of urban referral centers. We conducted a prospective observational single-center cohort study in May 2013 to assess the presentation, management and outcomes of adult and pediatric patients admitted with sepsis to a community hospital in rural Uganda.

Melioidosis, an infectious disease caused by the bacterium Burkholderia pseudomallei, is a common cause of sepsis in Southeast Asia. We investigated whether novel TLR1 coding variants are associated with outcome in Thai patients with melioidosis. We performed exonic sequencing on a discovery set of patients with extreme phenotypes (mild vs. severe) of bacteremic melioidosis. We analysed the association of missense variants in TLR1 with severe melioidosis in a by-gene analysis. We then genotyped key variants and tested the association with death in two additional sets of melioidosis patients. Using a by-gene analysis, TLR1 was associated with severe bacteremic melioidosis (P = 0.016). One of the eight TLR1 variants identified, rs76600635, a common variant in East Asians, was associated with in-hospital mortality in a replication set of melioidosis patients (adjusted odds ratio 1.71, 95% CI 1.01-2.88, P = 0.04.) In a validation set of patients, the point estimate of effect of the association of rs76600635 with 28-day mortality was similar but not statistically significant (adjusted odds ratio 1.81, 95% CI 0.96-3.44, P = 0.07). Restricting the validation set analysis to patients recruited in a comparable fashion to the discovery and replication sets, rs76600635 was significantly associated with 28-day mortality (adjusted odds ratio 3.88, 95% CI 1.43-10.56, P = 0.01). Exonic sequencing identifies TLR1 as a gene associated with a severe phenotype of bacteremic melioidosis. The TLR1 variant rs76600635, common in East Asian populations, may be associated with poor outcomes from melioidosis. This variant has not been previously associated with outcomes in sepsis and requires further study.

Staphylococcus argenteus is a newly named species previously described as a divergent lineage of Staphylococcus aureus that has recently been shown to have a global distribution. Despite growing evidence of the clinical importance of this species, knowledge about its population epidemiology and genomic architecture is limited. We used whole-genome sequencing to evaluate and compare S. aureus (n = 251) and S. argenteus (n = 68) isolates from adults with staphylococcal sepsis at several hospitals in northeastern Thailand between 2006 and 2013. The majority (82%) of the S. argenteus isolates were of multilocus sequence type 2250 (ST2250). S. aureus was more diverse, although 43% of the isolates belonged to ST121. Bayesian analysis suggested an S. argenteus ST2250 substitution rate of 4.66 (95% confidence interval [CI], 3.12 to 6.38) mutations per genome per year, which was comparable to the S. aureus ST121 substitution rate of 4.07 (95% CI, 2.61 to 5.55). S. argenteus ST2250 emerged in Thailand an estimated 15 years ago, which contrasts with the S. aureus ST1, ST88, and ST121 clades that emerged around 100 to 150 years ago. Comparison of S. argenteus ST2250 genomes from Thailand and a global collection indicated a single introduction into Thailand, followed by transmission to local and more distant countries in Southeast Asia and further afield. S. argenteus and S. aureus shared around half of their core gene repertoire, indicating a high level of divergence and providing strong support for their classification as separate species. Several gene clusters were present in ST2250 isolates but absent from the other S. argenteus and S. aureus study isolates. These included multiple exotoxins and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus, consistent with a livestock reservoir for S. argenteus These genes appeared to be associated with plasmids and mobile genetic elements and may have contributed to the biological success of ST2250.IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen.

Leptospirosis is a zoonotic infectious disease caused by pathogenic Leptospira species. Virulence proteins have been shown to be key determinants of the pathogenesis of pathogenic Leptospira. A specific peptide at a mass-to-charge ratio of 7000 Da was identified in Leptospira whole cells using matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. This peptide was specifically present in pathogenic Leptospira and in clinical isolates. We report here the characterization of this specific peptide using a proteomics approach. This peptide was significantly matched to a hypothetical conserved L. interrogans protein (LA2458) with a calculated molecular weight of 7140.136 Da containing a tellurite-resistance domain at its C terminus (TerB-C). The amino acid sequences revealed the presence of hydrophobic transmembrane portions and two linear B-cell epitopes. Despite its low abundance, this synthetic peptide demonstrated dose-dependent cytotoxicity toward African green monkey kidney (Vero) cells via the apoptosis pathway. The concentration of the peptide 100 µM induced about 50% of cell death after a 24 h exposure. This peptide could be useful for the diagnosis of leptospirosis and the study of pathogenesis.

Few studies of biomarkers as predictors of outcome in infection have been performed in tropical, low- and middle-income countries where the burden of sepsis is highest. We evaluated whether selected biomarkers could predict 28-day mortality in infected patients in rural Thailand.

Community acquired bacteremia (CAB) is a common cause of sepsis in low and middle-income countries (LMICs). However, knowledge about factors associated with outcomes of CAB in LMICs is limited.

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.

Critically ill patients manifest many of the same immune features seen in coronavirus disease 2019 (COVID-19), including both "cytokine storm" and "immune suppression." However, direct comparisons of molecular and cellular profiles between contemporaneously enrolled critically ill patients with and without severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited. We sought to identify immune signatures specifically enriched in critically ill patients with COVID-19 compared with patients without COVID-19. We enrolled a multisite prospective cohort of patients admitted under suspicion for COVID-19, who were then determined to be SARS-CoV-2-positive (n = 204) or -negative (n = 122). SARS-CoV-2-positive patients had higher plasma levels of CXCL10, sPD-L1, IFN-γ, CCL26, C-reactive protein (CRP), and TNF-α relative to SARS-CoV-2-negative patients adjusting for demographics and severity of illness (Bonferroni P value < 0.05). In contrast, the levels of IL-6, IL-8, IL-10, and IL-17A were not significantly different between the two groups. In SARS-CoV-2-positive patients, higher plasma levels of sPD-L1 and TNF-α were associated with fewer ventilator-free days (VFDs) and higher mortality rates (Bonferroni P value < 0.05). Lymphocyte chemoattractants such as CCL17 were associated with more severe respiratory failure in SARS-CoV-2-positive patients, but less severe respiratory failure in SARS-CoV-2-negative patients (P value for interaction < 0.01). Circulating T cells and monocytes from SARS-CoV-2-positive subjects were hyporesponsive to in vitro stimulation compared with SARS-CoV-2-negative subjects. Critically ill SARS-CoV-2-positive patients exhibit an immune signature of high interferon-induced lymphocyte chemoattractants (e.g., CXCL10 and CCL17) and immune cell hyporesponsiveness when directly compared with SARS-CoV-2-negative patients. This suggests a specific role for T-cell migration coupled with an immune-checkpoint regulatory response in COVID-19-related critical illness.