Hypervirulent Klebsiella pneumoniae (hvKP) causes Klebsiella‐induced liver abscess. Capsule is important for the pathogenesis of Klebsiella in systemic infection, but its role in gut colonisation is not well understood. By generating ΔwcaJ, Δwza and Δwzy capsule‐null mutants in a prototypical K1 hypervirulent isolate, we show that inactivation of wza (capsule exportase) and wzy (capsule polymerase) confer cell envelope defects in addition to capsule loss, making them susceptible to bile salts and detergent stress. Bile salt resistance is restored when the initial glycosyltransferase wcaJ was inactivated together with wzy, indicating that build‐up of capsule intermediates contribute to cell envelope defects. Mouse gut colonisation competition assays show that the capsule and its regulator RmpA were not required for hvKP to persist in the gut, although initial colonisation was decreased in the mutants. Both ΔrmpA and ΔwcaJ mutants gradually outcompeted the wild type in the gut, whereas Δwza and Δwzy mutants were less fit than wild type. Together, our results advise caution in using the right capsule‐null mutant for determination of capsule's role in bacterial pathogenesis. With the use of ΔwcaJ mutant, we found that although the capsule is important for bacterial survival outside the gut environment, it imposes a fitness cost in the gut.

T he global rise of carbapenem-resistant Enterobacterales (CRE) infections is posing a grave challenge to hospital systems worldwide (1). Carbapenemase genes usually are located on plasmids that can transmit vertically along clonal lineages and horizontally between different strains and species (2). However, the principles governing the transmission of carbapenemase-encoding plasmids in clinically relevant settings are complex and dynamic. Plasmid properties, donor, recipient, and ecologic factors all affect transmission (3,4).Previously, we found a 71,861-bp pKPC2 plasmid, pKPC2_sg1 (GenBank accession no. MN542377), in all 18 carbapenem-resistant hypervirulent Klebsiella pneumoniae isolates available in the Carbapenemase-Producing Enterobacteriaceae in Singapore (CaPES) collection (5,6). (Enterobacteriaceae is the former name of Enterobacterales.) The plasmid sequence was stable and unchanged after moving into different bacterial hosts or when maintained in human hosts for >200 days. This discovery prompted questions about the extent of pKPC2_sg1 dominance in clinical settings in Singapore, and its transmissibility and stability in hypervirulent K. pneumoniae. Using >1,000 CRE isolates collected from the 6 public hospitals in Singapore during 2010-2015, a subset of which was previously described (6), we examined the distribution of different carbapenem-encoding plasmids to investigate the dynamics and dominance of pKPC2. Materials and Methods Bacterial Strains, Growth Conditions, and PlasmidsWe analyzed 1,215 CRE isolates for carbapenemase plasmids distribution (Appendix 1 Table ,

Hypervirulent Klebsiella pneumoniae causes liver abscess and potentially devastating metastatic complications. The majority of Klebsiella -induced liver abscess are caused by the CG23-I sublineage of hypervirulent Klebsiella pneumoniae . This and some other lineages possess a >200-kb virulence plasmid. We discovered a novel protein IroP nestled in the virulence plasmid-encoded salmochelin operon that cross-regulates and suppresses the promoter activity of chromosomal type 3 fimbriae (T3F) gene transcription. IroP is itself repressed by iron through the ferric uptake regulator. Iron-rich conditions increase T3F and suppress capsule mucoviscosity, leading to biofilm formation and cell adhesion. Conversely, iron-poor conditions cause a transcriptional switch to hypermucoid capsule production and T3F repression. The likely acquisition of iroP on mobile genetic elements and successful adaptive integration into the genetic circuitry of a major lineage of hypervirulent K. pneumoniae reveal a powerful example of plasmid chromosomal cross talk that confers an evolutionary advantage. Our discovery also addresses the conundrum of how the hypermucoid capsule that impedes adhesion could be regulated to facilitate biofilm formation and colonization. The acquired ability of the bacteria to alternate between a state favoring dissemination and one that favors colonization in response to iron availability through transcriptional regulation offers novel insights into the evolutionary success of this pathogen. IMPORTANCE Hypervirulent Klebsiella pneumoniae contributes to the majority of monomicrobial-induced liver abscess infections that can lead to several other metastatic complications. The large virulence plasmid is highly stable in major lineages, suggesting that it provides survival benefits. We discovered a protein IroP encoded on the virulence plasmid that suppresses expression of the type 3 fimbriae. IroP itself is regulated by iron, and we showed that iron regulates hypermucoid capsule production while inversely regulating type 3 fimbriae expression through IroP. The acquisition and integration of this inverse transcriptional switch between fimbriae and capsule mucoviscosity shows an evolved sophisticated plasmid-chromosomal cross talk that changes the behavior of hypervirulent K. pneumoniae in response to a key nutrient that could contribute to the evolutionary success of this pathogen.