Abstract Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD largely enhanced liver fitness and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced, and DsbA, SspA, and PdxA increased fitness across bacteremia phases. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient ( Cybb −/− ) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis. Author Summary Gram-negative bacteremia is a deadly family of infections that initiate sepsis, a leading cause of global morbidity and mortality. Only a small number of Gram-negative species contribute to the majority of clinical bacteremia. Klebsiella pneumoniae is the second leading cause of Gram-negative bacteremia, and the third leading cause of overall bloodstream infection. K. pneumoniae is highly linked to hospital-associated infection with increasing antimicrobial resistance, endangering the most vulnerable patients. It is critical to understand the pathogenesis of K. pneumoniae bacteremia to better develop targets for future therapies that can prevent these deadly infections. Here, we define over 50 K. pneumoniae genes that support bloodstream fitness. These factors are diverse, support tissue-specific fitness, and increase bacterial resistance to oxidative stress. Our study is the first to systematically define K. pneumoniae factors enhancing bacteremia in a mammalian system. These results illuminate host-pathogen interactions during K. pneumoniae bacteremia that may be extended to additional Gram-negative species.

Klebsiella pneumoniaecauses bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress