Hypervirulent K. Pneumoniae Secretes More and More Active Iron-Acquisition Molecules than “Classical” K. Pneumoniae Thereby Enhancing its Virulence

A new hypervirulent (hypermucoviscous) clinical variant of Klebsiella pneumoniae (hvKP) has emerged over the last decade. Our goal is to identify new mechanisms, which increase the virulence hvKP compared to "classic" K. pneumoniae (cKP).Various growth assays were performed in human ascites, human serum, and laboratory medium with the hvKP strain hvKP1 (wt), randomly chosen blood isolates of cKP strains (cKP1-4), and mutant constructs deficient in the secretion of selected compounds. An in vivo mouse model that mimics infection due to hvKP and a quantitative siderophore assay were also used. It was established that a molecule(s)/factor(s) was secreted by hvKP1 significantly enhanced its growth and/or survival in human ascites. This molecule(s)/factor(s) also increased the growth and/or survival of hvKP1 in serum ex vivo and in an in vivo mouse model that measures metastatic spread after subcutaneous challenge, thereby further establishing biologic significance. Although features such as a size of <3kD, heat stability, and growth characteristics in ascites suggested this molecule(s) was a quorum-sensing compound, data presented demonstrates that this molecule(s)/factor(s) is involved in iron uptake and is likely a siderophore(s) or another iron-acquisition molecule. Although it is known that iron acquisition is critical for virulence, a novel aspect of this observation is that hvKP1 produces quantitatively more siderophores that appear to be biologically more active (increased affinity for iron or more resistant to host factors) than those produced by cKP strains.The data presented delineates a new mechanism by which hvKP increases its pathogenic potential compared to cKP strains. This paradigm may be broadly applicable to other extraintestinal gram-negative bacilli

The survival of hvKP1 in 80% human serum is significantly increased when serum is supplemented with hvKP1 generated conditioned ascites or minimal medium.

<p>The survival of hvKP1 in 80% active or heat inactivated (56°C for 30 minutes (Δ56°C)) human serum was assessed at 0, 3, 6, and 24 hours. Growth in heat inactivated serum served was a positive control that established that decreased survival was due to complement-mediated bactericidal activity. The active serum was supplemented with final concentrations of: 1) 20% 1×phosphate-buffered saline (PBS), 2) 15% PBS plus 5% unconditioned ascites (uncond-ascites; batch 2), 3) 10% PBS and 10% unconditioned M9 minimal medium (uncond-MM), 4) 15% PBS and 5% conditioned ascites (cond-ascites; batch 2), and 5) 10% PBS and 10% conditioned M9 minimal medium (cond-MM). The heat inactivated serum was supplemented with final concentrations of: 1) 15% PBS and 5% unconditioned ascites (uncond-ascites) and 2) 15% PBS and 5% conditioned ascites (cond-ascites). Survival of hvKP1 was significantly increased when 80% active serum was supplemented with hvKP1-conditioned ascites compared to unconditioned ascites (*P<0.05/1) or when supplemented with conditioned M9 minimal medium compared to unconditioned M9 minimal medium (*P<0.05/1). Survival of hvKP1 was significantly increased when 80% heat inactivated serum was supplemented with hvKP1-conditioned ascites compared to unconditioned ascites (*P<0.05/1). Data are Mean ± S.E.M.; N = 3–4.</p

The mediators present in hvKP1 generated conditioned minimal medium and ascites that enhance the growth and/or survival of hvKP1 are <3kD and heat stable.

<p>The growth of hvKP1 was assessed at 0, 3, 6, and 24 hours in human ascites (batch 2). <u>Panel A</u>: Ascites was supplemented with either hvKP1 generated conditioned (cond) M9 minimal medium (MM) (10% final concentration) that was: 1) non-fractionated (unfract), 2) <3kD fraction, and 3) >10kD fraction or unconditioned (uncond) minimal medium (10% final concentration) that was: 4) non-fractionated, and 5) <10kD fraction. The growth and/or survival of hvKP1 was significantly increased when the ascites was supplemented with 10% conditioned, non-fractionated minimal medium or with 10% conditioned, <3kD fraction of minimal medium compared to 10% unconditioned, non-fractionated minimal medium and 10% unconditioned, <10kD fraction of minimal medium respectively (* P<0.5/1). Data are Mean ± S.E.M.; N = 2-3. <u>Panel B</u>: Ascites was not supplemented (unconditioned) or supplemented with hvKP1 generated conditioned (cond) ascites or with hvKP1 generated conditioned ascites that was heated at 56°C for 30 minutes (Δ56°C) (batch 2) (5% final concentration for both supplements). The growth and/or survival of hvKP1 was significantly increased when ascites was supplemented with either conditioned or conditioned medium Δ56°C compared to unconditioned ascites (* P<0.05/2). Data are Mean ± S.E.M.; N = 4.</p

Growth and/or survival of hvKP1 grown in human ascites supplemented with varying concentration of homologous, conditioned ascites.

<p>The growth and/or survival of hvKP1 was assessed at 0, 3, 6, and 24 hours in 100% human ascites supplemented with homologous conditioned ascites resulting in final concentrations of 0%, 1%, 5%, 10%, and 25%. Growth and/or survival was also assessed in ascites supplemented with 25% unconditioned ascites heated at 56°C for 30 minutes to control for a possible effect of conditioned ascites diluting complement-mediated bactericidal activity. <u>Panel A</u>: ascites batch 1. <u>Panel B:</u> ascites batch 2. Supplementation with 5%, 10%, and 25% conditioned, homologous ascites, compared to 0%, resulted in a significant increase in growth and/or survival for both batches of ascites (* P<0.05/4) and supplementation with 1% conditioned, homologous ascites resulted in a trend for increased growth and/or survival (# P>0.05/4 and<0.05). Data are Mean ± S.E.M.; N = 2–3.</p

Neither LuxS generated AI-2 signaling molecules or the AI-3 signaling system appeared to affect the growth and/or survival of hvKP1 in conditioned ascites.

<p>The growth of hvKP1 was assessed at 0, 3, 6, and 24 hours in human ascites (batch 2). <u>Panel A</u>: Ascites was not supplemented (unconditioned) or supplemented with hvKP1 generated conditioned ascites (final concentration 5%) or hvKP1Δ<i>luxS</i> generated conditioned ascites (batch 2) (final concentration 5%). There was no significant difference in the growth of hvKP1 when supplemented with either of these conditioned media. <u>Panel B</u>: Ascites was not supplemented (unconditioned) or supplemented with hvKP1 generated conditioned ascites (batch 2) (final concentration 5%) or epinephrine (final concentrations 50 µM and 500 µM). There was no significant difference in the growth of hvKP1 with the addition of either of these supplements. Data are Mean ± S.E.M.; N = 2–3.</p

Siderophore concentration in hvKP1 and cKP1-4 generated conditioned ascites.

<p>*P<0.05/4 , hvKP1 compared to cKP1-4.</p

hvKP1 grown in conditioned ascites, compared to unconditioned ascites, resulted in significantly greater growth and/or survival and dissemination after SQ challenge of CD1 mice.

<p>Animals were challenged subcutaneously with 3.15×10⁴ cfu of hvKP1 grown in conditioned ascites (batch 2) and 1.89×10⁴ cfu or 1.65×10⁵ cfu of hvKP1 grown in unconditioned ascites (batch 2)(n = 6 for each condition and challenge inocula). Bacterial titers were enumerated in blood and various organs at 24 and 48 hours after challenge. After blood was obtained for culture, the vasculture was “flushed” with 1xPBS and organs were subsequently harvested. Comparisons were between hvKP1 grown in conditioned ascites and hvKP1 grown in unconditioned ascites. * P<0.05/2; # P>0.05/2 but <0.1. Data are Mean ± S.E.M. for n =  3.</p

hvKP1 generated conditioned ascites is a more potent enhancer of growth/survival in ascites of cKP1-4 than cKP1-4 generated conditioned ascites.

<p>The growth of cKP1-4 was assessed at 0, 3, 6, and 24 hours in human ascites (batch 2) that was heated at 56°C for 30 minutes (Δ56°C) and supplemented with 0%, 1%, 5%, or 25% of hvKP1 or cKP1-4 generated conditioned Δ56°C ascites. <u>Panel A</u>: cKP1; there was a trend for increased growth and/or survival of cKP1 when ascites was supplemented with 25% hvKP1-generated conditioned ascites compared to 25% cKP1-generated conditioned ascites (#P>0.05/3 but <0.05). <u>Panel B</u>: cKP2; the growth and/or survival of cKP2 was significantly increased when ascites was supplemented with 25% hvKP1-generated conditioned ascites compared to 25% cKP2-generated conditioned ascites (*P<0.05/3). <u>Panel C</u>: cKP3; the growth and/or survival of cKP3 was significantly increased when ascites was supplemented with 1% hvKP3-generated conditioned ascites compared to 1% cKP3-generated conditioned ascites (*P<0.05/3) and there was a trend for increased growth and/or survival of cKP1 when ascites was supplemented with 5% and 25% hvKP1-generated conditioned ascites compared to 5% and 25% cKP1-generated conditioned ascites (#P>0.05/3 but <0.05). <u>Panel D</u>: cKP4; there was a trend for increased growth and/or survival of cKP4 when ascites was supplemented with 1% hvKP1-generated conditioned ascites compared to 1% cKP4-generated conditioned ascites (#P>0.05/3 but<0.05). Data are Mean ± S.E.M.; N = 4.</p

Growth/survival of hvKP1 grown in human ascites supplemented with hvKP1 generated M9 and LB conditioned media.

<p>The growth of hvKP1 was assessed at 0, 3, 6, and 24 hours in human ascites (batch 2) supplemented with unconditioned (uncond) or hvKP1 generated conditioned (cond) rich laboratory (LB) or minimal (M9) media resulting in final concentrations of 0%, 1%, 5%, and 10%. <u>Panel A:</u> ascites supplemented with conditioned rich laboratory medium (LB). <u>Panel B</u>: ascites supplemented with conditioned minimal medium (MM). Supplementation with 10% conditioned MM compared to 0% resulted in a significant increase in growth/survival (* P<0.05/3) and supplementation with 5% conditioned MM compared to 0% resulted in a trend towards an increase in growth/survival (# P>0.05/3 and<0.05). Data are Mean ± S.E.M.; N = 2.</p

Growth and/or survival of hvKP1 (wt) in 100% human ascites and M9 minimal medium.

<p>The growth and/or survival of hvKP1 was assessed at 0, 3, 6, and 24 hours in 100% human ascites (batch 2) or M9 minimal medium at the starting inocula of approximately 10³ cfu/ml, 10⁵ cfu/ml, and 10⁷ cfu/ml. <u>Panel A</u>: 100% human ascites, Panel B: 100% human ascites heated at 56°C for 30 minutes, and <u>Panel C:</u> M9 minimal medium. Data are Mean ± S.E.M.; N = 3.</p