Multidrug resistance (MDR) represents a global threat to health. Here, we used whole genome sequencing to characterise Pseudomonas aeruginosa MDR clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicates that: 1) these large plasmids comprise an emerging family present in different members of the Pseudomonas genus, and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the accessory genome of the megaplasmid family is highly flexible and diverse. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.

Antibiotic resistance, particularly beta-lactam resistance, is a major problem worldwide. Imipenemase or IMP-type metallo-β-lactamase (MBL) has become a more prominent enzyme, especially in Asia, since it was discovered in the 1990s in Japan. There are currently 88 variants of IMP-type enzymes. The most commonly identified variant of IMP-type enzymes is IMP−1 variant. IMP-type MBLs have been detected in more than ten species in Enterobacterales. Pseudomonas aeruginosa is the most frequent carrier of IMP-type enzymes worldwide. In Asia, IMP-type MBLs have been distributed in many countries. This work investigated a variety of currently available IMP-type MBLs at both a global level and a regional level. Out of 88 variants of IMP-type MBLs reported worldwide, only 32 variants were found to have susceptibility profiles. Most of the bacterial isolates carrying IMP-type MBLs were resistant to Carbapenems, especially Imipenem and Meropenem, followed by the 3rd-generation cephalosporins, and interestingly, monobactams. Our results comprehensively indicated the distribution of IMP-type MBLs in Asia and raised the awareness of the situation of antimicrobial resistance in the region.

The bacterial genus Staphylococcus comprises diverse species with most being described as colonizers of human and animal skin. A relational analysis of features that discriminate its species and contribute to niche adaptation and survival remains to be fully described. In this study, an interspecies, whole-genome comparative analysis of 21 Staphylococcus species was performed based on their orthologues. Three well-defined multi-species groups were identified: group A (including aureus/epidermidis); group B (including saprophyticus/xylosus) and group C (including pseudintermedius/delphini). The machine learning algorithm Random Forest was applied to prioritize orthologs that drive formation of the Staphylococcus species groups A-C. Orthologues driving staphylococcal intrageneric diversity comprised regulatory, metabolic and antimicrobial resistance proteins. Notably, the BraSR (NsaRS) two-component system (TCS) and its associated BraDE transporters that regulate antimicrobial resistance showed limited distribution in the genus and their presence was most closely associated with a subset of Staphylococcus species dominated by those that colonize human skin. Divergence of BraSR and GraSR antimicrobial peptide survival TCS and their associated transporters was observed across the staphylococci, likely reflecting niche specific evolution of these TCS/transporters and their specificities for AMPs. Experimental evolution, with selection for resistance to the lantibiotic nisin, revealed multiple routes to resistance and differences in the selection outcomes of the BraSR-positive species S. hominis and S. aureus. Selection supported a role for GraSR in nisin survival responses of the BraSR-negative species S. saprophyticus. Our study reveals diversification of antimicrobial-sensing TCS across the staphylococci and hints at differential relationships between GraSR and BraSR in those species positive for both TCS.

Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection. Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured. iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated. iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future. Almost half of the world population is at risk of dengue viral infection. The disease severity varies from mild to a deadly form-which is caused mainly by host overt immune reaction. Earlier studies focused on the disease-causing roles of adaptive immune cells - cells that are highly specific but require time and signal from rapidly activating immune cells to become active. Invariant Natural Killer T (iNKT) cells are unique T cells that get activated rapidly and can control later adaptive response by secreting cytokines. They can be activated by lipid loaded on CD1d, an antigen presenting molecule. However, their role in human dengue infection was not known. Here, we studied iNKT cells from dengue infected children and found that they were activated. Importantly, the more severe the disease, the higher level of iNKT cells activation. Their cytokine patterns also differ from those of healthy donors. Moreover, together with iNKT cells activation, the level of CD1d was higher and T cells became active. Therefore, iNKT cells likely play a role in the pathogenesis of human dengue infection. New drugs targeting iNKT cells might help dampen the disease severity before the adaptive immune cells become too active.