Rapid design of bacteriophage cocktails to suppress the burden and virulence of gut-resident carbapenem-resistant Klebsiella pneumoniae

Antibiotic use can lead to expansion of multi-drug resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank that enables the rapid design of antimicrobial bacteriophage cocktails to treat multi-drug resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae, we identified host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank and experimental evolution strategies, we formulated combinations of phages that minimize the occurrence of phage resistance in vitro. Optimized bacteriophage cocktails selectively suppressed the burden of multi-drug resistant K. pneumoniae in the mouse gut microbiome and drove bacterial populations to lose key virulence factors that act as phage receptors. Further, phage-mediated diversification of bacterial populations in the gut enabled co-evolution of phage variants with higher virulence and a broader host range. Altogether, the Klebsiella PhageBank represents a roadmap for both phage researchers and clinicians to enable phage therapy against a critical multidrug-resistant human pathogen. Overall design: To further investigate the effect of csrD deletion on cell metabolism, we performed RNA-seq on MH258 and ?csrD. Cells were grown in vitro to mid-log phase for RNA isolation and RNA sequencing. Differential expression and gene set enrichment analysis was done to analyze how the loss of the carbon storage regulator (csrD) affects phage infection.