Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei, whose portals of entry into the body include subcutaneous, ingestion and inhalation routes. Animal models play an important role in furthering our understanding of this disease, which is associated with high morbidity and mortality in susceptible subjects. Previous studies using intranasal inoculation showed a differential susceptibility to inhalational melioidosis in BALB/c and C57Bl/6 mice and attributed the difference to genetic factors and host response. However, a recent study found no difference in susceptibility when the two species of mice were exposed to nebulized bacteria. We sought to address this discrepancy by using a nasal route only, instead of whole-body aerosol exposure system. Employing three different clinical strains of B. pseudomallei and following the progression of disease development in both BALB/c and C57Bl/6 mice, we found that BALB/c mice were at least 10- to 100-fold more susceptible to infection than C57Bl/6 mice. Comparison of bacterial burdens in aerosol-challenged mice, at both the pulmonary and distant sites of infection, suggests that C57Bl/6 mice were more efficient in clearing the bacteria than BALB/c mice. In addition, a comprehensive study of a wide panel of chemokines and cytokines at the protein level demonstrated that hyperproduction of proinflammatory cytokines in aerosol-challenged BALB/c mice did not translate into better protection and survival of these mice, whereas a moderate increase in these proteins in aerosol-challenged C57Bl/6 mice was more beneficial in clearing the infection. This suggests that high levels of proinflammatory cytokines are detrimental and contribute to the immunopathogenesis of the infection.

Background: 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. Results: Of 39 genomic regions variably present across the B. thailandensis strains, 13 regions corresponded to known genomic islands, while 26 regions were novel. Variant B. thailandensis isolates exhibited isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (B. pseudomallei-like capsular polysaccharide) closely resembling a similar cluster in B. pseudomallei that is essential for virulence in mammals; presence of this cluster was confirmed by whole genome sequencing of a representative variant strain (B. thailandensis E555). Both whole-genome microarray and multi-locus sequence typing analysis revealed that the variant strains formed part of a phylogenetic subgroup distinct from the ancestral B. thailandensis population and were associated with atypical isolation sources when compared to the majority of previously described B. thailandensis strains. In functional assays, B. thailandensis E555 exhibited several B. pseudomallei-like phenotypes, including colony wrinkling, resistance to human complement binding, and intracellular macrophage survival. However, in murine infection assays, B. thailandensis E555 did not exhibit enhanced virulence relative to other B. thailandensis strains, suggesting that additional factors are required to successfully colonize and infect mammals. Conclusions: The discovery of such novel variant strains demonstrates how unbiased genomic surveys of non-pathogenic isolates can reveal insights into the development and emergence of new pathogenic species.

Burkholderia pseudomallei , the etiological agent of melioidosis, is a facultative intracellular pathogen. As B. pseudomallei is a gram-negative bacterium, its outer membrane contains lipopolysaccharide (LPS) molecules, which have been shown to have low-level immunological activities in vitro. In this study, the biological activities of B. pseudomallei LPS were compared to those of Burkholderia thailandensis LPS, and it was found that both murine and human macrophages produced levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 in response to B. pseudomallei LPS that were lower than those in response to B. thailandensis LPS in vitro. In order to elucidate the molecular mechanisms underlying the low-level immunological activities of B. pseudomallei LPS, its lipid A moiety was characterized using mass spectrometry. The major lipid A species identified in B. pseudomallei consists of a biphosphorylated disaccharide backbone, which is modified with 4-amino-4-deoxy-arabinose (Ara4N) at both phosphates and penta-acylated with fatty acids (FA) C _14:0 (3-OH), C _16:0 (3-OH), and either C _14:0 or C _14:0 (2-OH). In contrast, the major lipid A species identified in B. thailandensis was a mixture of tetra- and penta-acylated structures with differing amounts of Ara4N and FA C _14:0 (3-OH). Lipid A species acylated with FA C _14:0 (2-OH) were unique to B. pseudomallei and not found in B. thailandensis . Our data thus indicate that B. pseudomallei synthesizes lipid A species with long-chain FA C _14:0 (2-OH) and Ara4N-modified phosphate groups, allowing it to evade innate immune recognition.

Melioidosis is a notifiable infectious disease registered with the Ministry of Health (MOH) and Agri-Food & Veterinary Authority (AVA), Singapore. From a clinical perspective, increased awareness of the disease has led to early detection and treatment initiation, thus resulting in decreasing mortality rates in recent years. However, the disease still poses a threat to local pet, zoo and farm animals, where early diagnosis is a challenge. The lack of routine environmental surveillance studies also makes prevention of the disease in animals difficult. To date, there have been no reports that provide a complete picture of how the disease impacts the local human and animal populations in Singapore. Information on the distribution of Burkholderia pseudomallei in the environment is also lacking. The aim of this review is to provide a comprehensive overview of both published and unpublished clinical, veterinary and environmental studies on melioidosis in Singapore to achieve better awareness and management of the disease.

Introduction: Since the outbreak of the COVID-19 pandemic, extensive efforts including vaccine and drug development have been accentuated to address the emergence of SARS-CoV-2 variants. The rising variants and subvariants made early discoveries in effective treatment strategies less relevant and potent. For instance, multiple therapeutics like Evusheld are no longer effective against current variants and therefore have their emergency use authorizations (EUA), a regulatory mechanism allowing emergency use of medical products, revoked until further notice. Similarly, sotrovimab (STV), a human neutralizing monoclonal antibody (MAB), received EUA in May 2021 and was later granted authorization for COVID-19 treatment in Singapore. However, mutations presented in XBB, a subvariant of Omicron, substantially reduced the potency of STV at the currently approved dose. Methods: To retain STV as a part of the treatment strategy for COVID-19 infected patients, it is important to carefully design and prioritize drugs that may further enhance the potency of STV when paired in combinations. In this study, IDentif.AI, an artificial intelligence (AI)-derived drug combination optimization platform, was harnessed to rapidly pinpoint STV-based combinations that may substantially improve the efficacy and potency of STV against XBB, which was predominantly spreading worldwide in 2023. Results: IDentif.AI-pinpointed STV/EIDD-1931 and STV/GS-441524 combinations were able to interact synergistically to enhance efficacy against XBB and importantly, substantially reduce STV's in vitro EC50 (half maximal effective concentration) by up to 7-fold. Discussion: This optimization platform represents a strategic approach to rapidly repurpose existing or previously failed therapeutics and subsequently restore their efficacy against a disease indication by pairing them with the correct drugs in combinations.