Extracellular polysaccharide production by Klebsiella pneumoniae and its relationship to virulence. PHILIP DOMENICO, DANA L. DIEDRICH, AND DAVID C.
Extracellular polysaccharide production by Klebsiella pneumoniae and its relationship to virulence PHILIP DOMENICO, DANAL. D IEDRICH, AND D AVID C. STRAUS' Department of Microbiology, Texas Tech Universiw Health Sciences Center, Lubbock, ïX, U.S.A. 79430 Accepted February 7, 1985 DOMENICO, P., D. L. DIEDRICH, and D. C. STRAUS. 1985. Extracellular polysaccharide production by Klebsiellapneumoniae and its relationship to virulence. Can. J. Microbiol. 31: 472-478. ~lebsiellapneumoniaeserotype 1 and serotype 2 and their capsular variants were examined for production of cell-associated capsular polysaccharides and extracellular capsular polysaccharides. The virulence of these organisms in experimental animals was examined via intraperitoneal injection in mice and transtracheal inoculation into the lungs of rats. It was found that the production of either polysaccharide component correlated with the obsemed virulence. The extracellular polysaccharides were purified by ethanol precipitation, electrodialysis, extraction with quaternary ammonium salts, and gel. filtration. These purification steps allowed for the separation and purification of both the extracellular lipopolysaccharide and the extracellular capsular polysaccharide. Purified extracellular capsular polysaccharide and extracellular lipopolysaccharide were CO-injected with K . pneumoniae intraperitoneally into mice to determine if either of these substances would produce an effect on the natural course of infection in these animals. These studies showed that only purified extracellular lipopolysaccharide enhanced the virulence of K . pneumoniae when CO-injectedinto mice, and this virulence enhancement correlated with the content of extracellular lipopolysaccharide, but not extracellular capsular polysaccharide in mixtures of these polysaccharides. Saponification of K . pneumoniae serotype 1 extracellular polysaccharides significantly decreased their virulence-enhancing capabilities in mice, further suggesting that extracellular lipopolysaccharide may play a role in these infections. et D. C. STRAUS. 1985. Extracellular polysaccharide production by Klebsiella pneumoniae DOMENICO, P., D. L. DIEDR~CH and its relationship to virulence. Can. J. Microbiol. 31: 472-478. On a étudié la production des polysaccharides capsulaires extracellulaires et des polysaccharides liés à la cellule chez Klebsiella pneumoniae sérotype 1 et sérotype 2 et chez des variants capsulaires. La virulence des souches a été évaluée dans des modèles animaux soit par inoculation intrapéritonéale chez la souris et inoculation dans le poumon par voie transtrachéale chez le rat. On a noté une corrélation entre le degré de virulence et la production de l'un ou l'autre des polysaccharides. Les polysaccharides extracellulaires ont été purifiés par précipitation à l'éthanol, électrodialyse, extraction avec des sels d'ammonium quaternaire et filtration sur gel. Cette séquence de purification a permis de séparer et de purifier le lipopolysaccharide extracellulaire et le polysaccharide capsulaire extracellulaire. Ce polysaccharide capsulaire extracellulaire purifié ainsi que le lipopolysaccharide extracellulaire ont été injectés par voie intrapéritonéale chez la souris en même temps qu'une souche de K . pneumoniae dans le but de vérifier si l'une ou l'autre de ces substances pouvait influencer le déroulement normal de l'infection. Ces essais ont démontré que seul le lipopolysaccharide extracellulaire purifié augmentait la virulence de K . pneumoniae et qu'il y avait une corrélation entre cette augmentation de virulence et la composition du lipopolysaccharide extracellulaire mais pas avec le polysaccharide capsulaire extracellulaire dans des mélanges de ces polysaccharides. Chez K . pneumoniae, sérotype 1, la saponification des polysaccharides extracellulaires diminuait significativement leur capacité d'augmenter la virulence chez la souris, suggérant ainsi que les lipopolysaccharides extracellulaires pourraient jouer un rôle dans ces infections. [Traduit par le journal]
Introduction It is well established that the polysaccharide capsule of Klebsiella pneumoniae is essential for the virulence of this bacterium to be expressed (Ehrenworth and Baer 1956; Julianelle 1926). We have suggested in a recent study employing a rat lung model, that the antiphagocytic properties of the capsule increase as the cell-associated capsular polysaccharides increase in volume (Domenico et al. 1982). Recently, however, Mizuta et al. (1983) found avirulent strains of Klebsiella that were fully encapsulated. Of nine strains belonging to Klebsiella serogroup 01 :K2, two were avirulent for mice, in contrast to the seven other strains with mean lethal dose (LD-j,) values of less than 10colony-forming units (CFU). These studies suggest that there is something other than cell-associated capsular material that determines the virulence of K . pneumoniae. We therefore decided to examine the role that the extracellular polysaccharides of K . pneumoniae might play in the pathogenesis of infections produced by this organism. Klebsiella pneumoniae is known to excrete large amounts of these materials in vitro (Ehrenworth and Baer 1956; Mizuta et al. 1983) and in vivo (Pollack 1976), but little is known about the significance of this phenomenon in the disease process. It was 'Author to whom al1 correspondence should be addressed.
shown previously (Ehrenworth and Baer 1956) that the cellfree capsular material elaborated by K . pneumoniae increases in proportion to the size of the cell-associated capsule and that these two parameters are probably different manifestations of the same phenomenon, i.e., the total rate of polysaccharide production. To what extent each is responsible for the increased pathogenicity of encapsulated organisms is, as of yet, unclear. Previous studies attempting to address these questions have been difficult to interpret since the capsular polysaccharides have not been shown to be completely separated from contaminating lipopolysaccharides. In this report, a number of K . pneumoniae strains were examined for polysaccharide production and for vidence in two rodent models. In addition, both the extracellular capsular polysaccharide (ECPS) and extracellular lipopolysaccharide (ELPS) were purified and coinjected with viable organisms into mice to examine the effect(s) that these substances might have on the normal course of infection.
Materials and methods Bacteria The Klebsiella pneumoniae serotype 1 (KPI) strains included ATCC 8047 (a lung isolate from the American Type Culture Collection, Rockville, MD) and CDC 2-70 from the Centers for Disease Control,
DOMENICO ET AL.
TABLE 1. Comparison of ECPS, ELPS, capsule size, and virulence of K . pneumoniae serotypes 1 and 2 Capsule diameter ( ~ m ) Organism
ECPS"
KPI LC KPI SC KPI 2-70 KP2 LC KP2 SC KP2 2-70
20.12 2.78 3.73 1.30 0.01 2.13
ELPS~ Mean 1.43 0.38 0.54 0.03 0.01 0.07
5.6 2.5 2.2 2.5 1.5 3.0
Range 3.9- 8.6 2.2- 3.0 1.8-2.5 1.9-3.0 1.4-1.6 2.5-5.0
LDso (CFU)'
lDsa (CFU)'
4.99~10'~ 4.31~ 5.34~10'~ 1 . 5 3 ~ 1 0 ~ ~ 7.30~10~ NP 1.78x1OSd >7.30x107 > 6 . 2 0 ~ 1 0 ~ ~ NP
