Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress.

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 supported liver fitness in vivo 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. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, 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.

The Klebsiella pneumoniae ter Operon Enhances Stress Tolerance.

Healthcare-acquired infections are a leading cause of disease in patients that are hospitalized or in long-term-care facilities. Klebsiella pneumoniae (Kp) is a leading cause of bacteremia, pneumonia, and urinary tract infections in these settings. Previous studies have established that the ter operon, a genetic locus that confers tellurite oxide (K2TeO3) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, the ter operon increases Kp fitness during gut colonization; however, the biologically relevant function of this operon is unknown. First, using a murine model of urinary tract infection, we demonstrate a novel role for the ter operon protein TerC as a bladder fitness factor. To further characterize TerC, we explored a variety of functions, including resistance to metal-induced stress, resistance to radical oxygen species-induced stress, and growth on specific sugars, all of which were independent of TerC. Then, using well-defined experimental guidelines, we determined that TerC is necessary for tolerance to ofloxacin, polymyxin B, and cetylpyridinium chloride. We used an ordered transposon library constructed in a Kp strain lacking the ter operon to identify the genes that are required to resist K2TeO3-induced and polymyxin B-induced stress, which suggested that K2TeO3-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K2TeO3 disrupts the Kp cell envelope, though these effects are independent of ter. Collectively, the results from these studies indicate a novel role for the ter operon as a stress tolerance factor, thereby explaining its role in enhancing fitness in the gut and bladder.

Combined comparative genomics and clinical modeling reveals plasmid-encoded genes are independently associated with Klebsiella infection.

Members of the Klebsiella pneumoniae species complex frequently colonize the gut and colonization is associated with subsequent infection. To identify genes associated with progression from colonization to infection, we undertook a case-control comparative genomics study. Concordant cases (N = 85), where colonizing and invasive isolates were identical strain types, were matched to asymptomatically colonizing controls (N = 160). Thirty-seven genes are associated with infection, 27 of which remain significant following adjustment for patient variables and bacterial phylogeny. Infection-associated genes are not previously characterized virulence factors, but instead a diverse group of stress resistance, regulatory and antibiotic resistance genes, despite careful adjustment for antibiotic exposure. Many genes are plasmid borne, and for some, the relationship with infection is mediated by gut dominance. Five genes were validated in a geographically-independent cohort of colonized patients. This study identifies several genes reproducibly associated with progression to infection in patients colonized by diverse Klebsiella.

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor.

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.