Fitness of gram-negative pathogens during bacteremia

This research addresses a critical and growing clinical problem: antibiotic-resistant Gram-negative bacterial infections in hospital and clinical settings. The project responds to alarming public health data describing more than two million annual infections with antibiotic-resistant bacterial pathogens and approximately 23,000 deaths each year, with over half of the problematic species being Gram-negative organisms such as carbapenem-resistant Enterobacteriaceae and Acinetobacter baumannii. Despite the clinical importance of these pathogens, the molecular determinants that enable virulence and in vivo survival during bloodstream infection remain poorly understood. This research aims to identify both species-specific and shared fitness and virulence factors required for bacteremia across six representative Gram-negative pathogens: Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, and Acinetobacter baumannii.

The project integrates high-throughput genetic and transcriptomic approaches with in vivo growth measurements in a murine model of bacteremia. Having largely completed Tn-seq for the selected isolates, the investigators will conduct RNA-seq to measure gene expression patterns during infection and quantify in vivo growth rates, enabling direct comparison of active metabolic pathways and expression profiles at equivalent growth phases across pathogens. The central objectives are to delineate core orthologous functions shared by related species and to discover unique pathways or genes required for nutrient acquisition, immune evasion, and replication in the bloodstream. By mapping metabolic pathways and annotating them with genes that are pathogen-specific or conserved across multiple species, the study seeks to reveal mechanistic insights into pathogenesis and to identify potential targets for therapeutic intervention.

Experimental outputs will include growth kinetics for each pathogen during bacteremia, lists of preferred and required metabolic pathways under in vivo conditions, and annotated pathway maps linking genetic determinants to functional processes critical for survival in the host bloodstream. Imaging and staining methods, such as Congo Red and Maneval stains, are utilized to visualize phenotypic features like capsule polysaccharide in representative organisms. Quantitative sequencing analyses, exemplified by read distributions across the E. coli CFT073 genome under varying nutrient conditions, support the effort to correlate environmental nutrient availability with pathogen genomic activity.

The investigation emphasizes comparative pathogen biology to identify both conserved vulnerabilities and species-specific adaptations that enable Gram-negative bacteria to cause life-threatening bacteremia. By characterizing active metabolic pathways, essential gene sets, and in vivo growth behavior across multiple clinically relevant species, the research aims to fill major gaps in understanding bloodstream infection biology and to inform strategies for combating antibiotic-resistant Gram-negative pathogens in healthcare settings.