In Vivo -Validated Essential Genes Identified in Acinetobacter baumannii by Using Human Ascites Overlap Poorly with Essential Genes Detected on Laboratory Media

Abstract

ABSTRACT A critical feature of a potential antimicrobial target is the characteristic of being essential for growth and survival during host infection. For bacteria, genome-wide essentiality screens are usually performed on rich laboratory media. This study addressed whether genes detected in that manner were optimal for the identification of antimicrobial targets since the in vivo milieu is fundamentally different. Mutant derivatives of a clinical isolate of Acinetobacter baumannii were screened for growth on human ascites, an ex vivo medium that reflects the infection environment. A subset of 34 mutants with unique gene disruptions that demonstrated little to no growth on ascites underwent evaluation in a rat subcutaneous abscess model, establishing 18 (53%) of these genes as in vivo essential. The putative gene products all had annotated biological functions, represented unrecognized or underexploited antimicrobial targets, and could be grouped into five functional categories: metabolic, two-component signaling systems, DNA/RNA synthesis and regulation, protein transport, and structural. These A. baumannii in vivo essential genes overlapped poorly with the sets of essential genes from other Gram-negative bacteria catalogued in the Database of Essential Genes (DEG), including those of Acinetobacter baylyi , a closely related species. However, this finding was not due to the absence of orthologs. None of the 18 in vivo essential genes identified in this study, or their putative gene products, were targets of FDA-approved drugs or drugs in the developmental pipeline, indicating that a significant portion of the available target space within pathogenic Gram-negative bacteria is currently neglected. IMPORTANCE The human pathogen Acinetobacter baumannii is of increasing clinical importance, and a growing proportion of isolates are multiantimicrobial-resistant, pan-antimicrobial-resistant, or extremely resistant strains. This scenario is reflective of the general problem of a critical lack of antimicrobials effective against antimicrobial-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa , Klebsiella pneumoniae , Enterobacter sp., and Escherichia coli . This study identified a set of A. baumannii genes that are essential for growth and survival during infection and demonstrated the importance of using clinically relevant media and in vivo validation while screening for essential genes for the purpose of developing new antimicrobials. Furthermore, it established that if a gene is absent from the Database of Essential Genes, it should not be excluded as a potential antimicrobial target. Lastly, a new set of high-value potential antimicrobial targets for pathogenic Gram-negative bacteria has been identified.