The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association

Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces

Seroepidemiology of Klebsiella pneumoniae in an Australian Tertiary Hospital and Its Implications for Vaccine Development

The aim of this study was to determine the diversity of Klebsiella pneumoniae capsular serotypes in an Australian setting. Consecutive (n = 293) nonrepetitive isolates of K. pneumoniae from a large teaching hospital laboratory were analyzed. The majority of isolates were from urinary specimens (60.8%); the next most common source was sputum (14.3%), followed by blood (14%). Serotyping revealed a wide range of capsule types. K54 (17.1%), K28 (4.1%), and K17 (3.1%) were the most common, and K54 isolates displayed a high degree of clonality, suggesting a common, nosocomial source. In vitro, one K54 isolate was more adherent to urinary catheters and HEp-2 cells than four other tested isolates; it was slightly more resistant to chlorhexidine but was more susceptible to drying than heavily encapsulated strains. This is the first seroprevalence survey of K. pneumoniae to be performed on Australian isolates, and the high level of diversity of serotypes suggests that capsule-based immunoprophylaxis might not be useful for Australia. In addition there are significant differences in the predominance of specific serotypes compared to the results of surveys performed overseas, which has important implications for capsule-based immunoprophylaxis aimed at a global market

Resistance of <i>K. pneumoniae</i> strains to complement mediated lysis.

*<p>HTS = Heat treated sera. Serum complement factors were inactivated by heating at 56°C for 30 min.</p><p>Note: A bacteriostatic antibiotic - tetracycline (25 µg/ml) - was added to samples to inhibit growth and hence allow direct measurement of bacterial killing by complement factors.</p><p>Figures are the average of duplicate samples, and the assay has been repeated in the presence and absence of tetracycline.</p

<i>K. pneumoniae</i> CPS and LPS saccharides affect the electrophoretic mobility of the bacteria.

<p>The electrophoretic mobility of B5055, B5055nm, B5055nm<i>waaF</i> and B5055nm<i>waaF</i> (pBR<i>waaF</i>) were compared in different ionic strength potassium nitrate buffers at the natural pH of the buffer (pH 5.5–6). Shown are the mean and SD of 60 trajectories for each strain, under each condition. The assay was repeated on different days with fresh cultures.</p

Characterisation of the surface layers of <i>K. pneumoniae</i> mutant strains.

<p>A) The capsule was visualised by Maneval's stain and quantified by uronic acid assay. UD = undetectable. B) LPS preparations were separated by SDS-PAGE on a 15% polyacrylamide gel and visualised by modified silver staining. C) Fluorescent activated cell sorting using mAb C13 specific for the O1-Ag of LPS. Only single cell populations (determined from cells stained with secondary antibody alone) were included in fluorescence analsyis. D) OMP preparations from mid-log cultures separated by 12.5% SDS-PAGE and visualised by Coomassie Brilliant Blue staining. Lanes are (1) B5055, (2) B5055nm, (3) B5055nm<i>waaF</i> and (4) B5055nm<i>waaF</i>+pBR<i>waaF</i>.</p

Association of <i>K. pneumoniae</i> mutant strains to mammalian cells.

<p>Bacteria were cultured to mid-log growth phase, washed, and added to confluent monolayers of RAW264.7 macrophage-like cells (A) or A549 epithelial cells (B). After 2 hrs, non-adherent bacteria were removed by repeated washing, cells were treated with Triton X-100 and cell lysates were plated for CFU enumeration. The number of cell associated bacteria are presented as the percentage of the initial inoculum. The data are the mean and standard deviation (SD) of 6 wells/strain and the assay was repeated at least three times.</p

Theoretical description of the experimental electrophoretic mobilities of <i>K. pneumoniae</i> strains.

<p>The electrophoretic mobility (filled circles) are graphed for A) B5055nm, B) B5055 and C) B5055nm<i>waaF K. pneumoniae</i> strains expressed as a function of ionic strength. The calculations were performed either by using a homogeneous description for the spatial distribution of the soft-permeable polymer (α = 0, dash lines) or using a heterogeneous distribution (α>0, dotted lines) to reproduce the experimental data over the whole range of ionic strength considered. The different parameters obtained from the best fitting procedure are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003817#pone-0003817-t001" target="_blank">Table 1</a>.</p