The Bhattacharyya lab focuses on characterizing the response of pathogens (bacteria and fungi) to antimicrobials, and of patients to systemic infections, with an emphasis on deepening our mechanistic understanding and diagnosis of infections. While we use many approaches to address these broad topics, our mainstays are a combination of standard microbiological methods and transcriptional profiling (RNAseq and scRNAseq). As an early manifestation of cellular responses to stimuli, gene expression profiles, particularly in response to key perturbations like antibiotic exposure or infection, reflect physiological adaptations by both microbes and patients that we can use to infer mechanisms of disease pathogenesis, and also co-opt for diagnostic purposes. Specific ongoing projects include:

1. A novel transcriptional approach to clinical antimicrobial susceptibility testing (AST): Antimicrobial resistance (AMR) is one of the most important medical challenges of our time. Yet diagnosis of AMR remains too slow to inform clinical care in real time, feeding an unsustainable cycle of ever-broadening empiric antibiotic use. We developed a novel approach to AST based on the observation that susceptible strains exhibit major transcriptional changes upon antibiotic exposure, while resistant strains do not, agnostic to resistance mechanism. We use RNA-Seq to identify transcripts whose antibiotic-induced expression best distinguishes susceptible from resistant strains, then measure them, along with key genotypic resistance elements, using a simple, rapid, robust multiplexed RNA detection platform (NanoString). Beyond pilot implementation studies, we are extending this approach to novel pathogen-antimicrobial pairs, including fungi, and also exploring these rich transcriptional datasets to better understand mechanisms of bacterial killing by, and adaptation to, key antibiotic classes.

2. Single-cell transcriptional profiling of circulating immune cells in patients with systemic infection: Better understanding the immune response to infection is crucial for life-threatening conditions such as sepsis and COVID-19, in which a dysregulated immune response contributes to pathogenesis. With collaborators from clinical medicine, immunology, and biotechnology (Drs. Mike Filbin, Marcia Goldberg, Nir Hacohen, and Paul Blainey), we used scRNAseq of peripheral blood mononuclear cells (PBMCs) to identify a novel transcriptional substate of monocytes (monocyte substate 1, or MS1) that is enriched in patients with sepsis and other causes of critical illness. Analysis of publicly available scRNAseq data reveals that MS1 is also enriched in severe COVID-19 and predictive of mortality. In an actively enrolling patient cohort, we are exploring the kinetics of the immune response to sepsis and non-infectious critical illness, and investigating heterogeneity among sepsis patients that may predict clinical outcomes and guide immunomodulatory therapeutic strategies. The gene expression signatures of MS1 suggest an immunosuppressive phenotype, but the role of MS1 in the pathophysiology of sepsis remains uncertain. Through transcriptional and functional studies, we are eager to explore how MS1 impacts the immune dysregulation that contributes to sepsis pathophysiology.

3. Elucidating mechanisms of carbapenem resistance: Carbapenems, in the beta-lactam class, are the broadest-spectrum antibiotics in clinical use, yet resistance is growing. Two major resistance mechanisms are known: (a) hydrolysis via carbapenemases, or (b) reduced access to their periplasmic targets, typically via disruptions in outer membrane porins, often in combination with lower-level hydrolysis by extended-spectrum beta-lactamases. While these two mechanisms, alone or together, account for most carbapenem resistance clinically encountered among Enterobacterales, some remains unexplained. Through in vitro evolution of clinical isolates with unexplained resistance and subsequent complementation studies, we uncovered a transcription factor not previously known to affect carbapenem resistance that clearly contributes to high-level resistance. We are currently working to understand its mechanisms of resistance, regulation, and fitness tradeoffs in the absence of antibiotics. This transcription factor has several homologs that function in stress responses, pointing to a broader role for intrinsic resistance factors in carbapenem resistance that we are eager to explore.