Integrative metabolomics and transcriptomics identifies itaconate as an adjunct therapy to treat ocular bacterial infection Graphical abstract Highlights d Bacterial infection increases Irg1 and itaconate levels in the eye d Irg1 and Nrf2 deficiency exacerbates intraocular bacterial infection d Itaconate potentiates antioxidant NRF2/HO-1 signaling in the eye d Itaconate treatment synergizes with antibiotics in ameliorating ocular infection

To evaluate the clinical utility of high-throughput sequencing (HTS) approach-based analysis of the bacterial and fungal genome in vitreous fluids from patients clinically diagnosed as endophthalmitis, we subjected 75 vitreous fluids from clinically presumed infectious endophthalmitis patients to high-throughput sequencing (Illumina HiSeq 2500) after DNA extraction and amplification of the 16S rRNA for the detection of bacteria, and ITS 2 region for detection of fungal pathogens. As controls, we included vitreous biopsies from 70 patients diagnosed with other non-infectious retinal disorders. Following the construction of the curated microbial genome database and filtering steps to reduce ambiguousness/contaminants from the environment, the paired reads were analyzed. Our HTS reads revealed in almost all cases the same organism that was grown in culture (bacterial-14/15, fungal 3/3) by conventional microbiological workup. HTS additionally diagnosed the presence of microbes in 42/57 (73.7%) patients who were conventionally negative (fungal pathogens in 36/57, bacterial pathogens in 11/57, including five cases that showed the presence of both bacterial and fungal organisms). Aspergillus sp., Fusarium sp., Exserohilum sp., and Candida sp. were the most predominant genera in our cohort of culture-negative endophthalmitis cases. Heat map based microbial clustering analysis revealed that these organisms were taxonomically similar to the species identified by conventional culture methods. Interestingly, 4/70 control samples also showed the presence of bacterial reads, although their clinical significance is uncertain. HTS is useful in detecting pathogens in endophthalmitis cases that elude conventional attempts at diagnosis and can provide actionable information relevant to management, especially where there is a high index of suspicion of fungal endophthalmitis, particularly in tropical countries. Outcome analyses and clinical trials addressing the success and cost savings of HTS for the diagnosis of endophthalmitis are recommended.

The alarming situation in public healthcare caused by ever‐increasing catastrophe of antimicrobial resistance, recurrent infections, and associated inflammation has accelerated the hunt for novel therapeutics which can address these diverse problems concomitantly. This article introduces a new class of multi‐functional amino acid conjugated small antibacterial molecules (ASAMs) which tackle complicated infections and associated inflammation. These molecules exhibit broad‐spectrum bactericidal activity against multi‐drug‐resistant bacteria. The phenylalanine‐bearing lead molecule (ASAM‐10) tackles bacterial dormant subpopulations, impenetrable biofilms, and intracellular pathogens simultaneously. Importantly, this molecule addresses the problem of toxicity associated with cationic lipopeptides like colistin through the temporal charge switching (cationic to zwitterionic) owing to the degradation of labile ester linkages. However, this does not affect its desired antibacterial action window. The substantial reduction in the overexpression of pro‐inflammatory cytokines (IL‐6, IL‐8, TNF‐α, and IL‐1β) upon treatment of infected macrophages with ASAM‐10 validates its anti‐inflammatory efficacy. Furthermore, bacteria exhibit diminished susceptibility toward resistance development against ASAM‐10 owing to its membrane‐active nature. ASAM‐10 displays significant reduction in bacterial burden (2 Log CFU/g) when administered intraperitoneally in mice for MRSA thigh infection. Overall, this new class of multi‐functional molecules is safe for anticipated advanced therapeutic applications to combat complex bacterial infections and inflammation.

Increasing incidences of multidrug-resistant (MDR) pathogens causing endophthalmitis threaten our ability to treat this condition, and the modulation of inflammatory responses by MDR bacteria is not known. In this study, using human microglia and retinal pigment epithelial (RPE) cells, we compare the inflammatory responses of sensitive (S-PA) and multidrug-resistant (MDR-PA) clinical isolates of Pseudomonas aeruginosa. Infected cells were subjected to qPCR analysis, enzyme-linked immunosorbent assay (ELISA), and immunostaining to assess the expression of inflammatory mediators. Both microglia and RPE cells, challenged with S-PA and MDR-PA, induced a time-dependent expression of inflammatory cytokines. Significant differences were observed in expression levels of Toll-like receptors (TLR) TLR4, TLR5, and TLR9 in microglia cells challenged with MDR-PA vs. S-PA. Similarly, mRNA levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, Interferon (IFN)-γ, and matrix metalloproteinase (MMP)-9 were also higher in MDR-PA-infected cells. At protein levels, upregulation was observed for IL-10 (p = 0.004), IL-8 (p = 0.0006), IL-1β (p = 0.02), and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (p = 0.0006) in cells infected MDR-PA versus S-PA in both microglia and RPE cells; however, the response was delayed in RPE cells. Heatmap and STRING analysis highlighted the existence of a cross-talk between the inflammatory and cytokine-mediated signaling pathways. Our study highlights a differential inflammatory response evoked by MDR vs. sensitive pathogens in retinal cells during endophthalmitis.