In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiella pneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost. Highly invasive strains of Klebsiella pneumoniae that exhibit the hypermucoviscosity/hypermucoviscous (HV) phenotype have been reported as human pathogens, causing liver abscesses in patients in America, Europe, South Korea, and Taiwan (1, 2). The HV phenotype of K. pneumoniae is due to capsular polysaccharide (CPS) production and the presence of virulence genes, such as magA, g-rmpA, p-rmpA, and p-rmpA2 (3, 4). Several studies demonstrated that the sequence type 23 (ST23) clone was associated with the K1 capsular serotype in HV K. pneumoniae strains that were isolated from liver abscesses (3). Meanwhile, the K2 serotype was reported for ST86, ST375, and ST380 HV isolates (2).Colistin (also known as polymyxin E) is an antimicrobial polypeptide that mediates bactericidal activity by interacting with the lipid A component of lipopolysaccharide (LPS) present on Gramnegative pathogens, including K. pneumoniae, leading to outer membrane disruption (5). It is known that the modification of LPS following the addition of 4-amino-4-deoxy-L-arabinose to lipid A is related to colistin resistance in K. pneumoniae. The modification of LPS is associated with the pbgPE operon, which is regulated by two-component regulatory systems, such as PmrAB and PhoPQ. Insertional inactivation of MgrB, a negative reg...

Klebsiella pneumoniae is a common cause of sepsis and is particularly associated with healthcare-associated infections. New strategies are needed to prevent or treat infections due to the emergence of multi-drug resistant K. pneumoniae. The goal of this study was to determine the diversity and distribution of O (lipopolysaccharide) and K (capsular polysaccharide) antigens on a large (>500) global collection of K. pneumoniae strains isolated from blood to inform vaccine development efforts. A total of 645

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are important human pathogens that are associated with a range of infection types, including wound and disseminated infections. Treatment has been complicated by rising rates of antimicrobial resistance. Immunoprophylactic strategies are not constrained by antimicrobial resistance mechanisms. Vaccines against these organisms would be important public health tools, yet they are not available. KP surface O polysaccharides (OPS) are protective antigens in animal models of infection. Similarly, PA flagellin (Fla), the major subunit of the flagellar filament, is required for virulence and is a target of protective antibodies in animal models. We report herein the development of a combined KP and PA glycoconjugate vaccine comprised of the four most common KP OPS types associated with human infections (O1, O2, O3, O5), chemically linked to the two Fla types of PA (FlaA, FlaB). Conjugation of KP OPS to PA Fla enhanced anti-polysaccharide immune responses and produced a formulation that generated antibody titers to the four KP OPS types and both PA Fla antigens in rabbits. Passive transfer of vaccine-induced rabbit antisera reduced the bacterial burden and protected mice against fatal intravenous KP infection. Mice passively transferred with conjugate-induced antisera were also protected against PA infection after thermal injury with a FlaB-expressing isolate, but not a FlaA isolate. Taken together, these promising preclinical results provide important proof-of-concept for a broad spectrum human vaccine to prevent KP and PA infections.