The different mechanisms of Kiebsiella pneumoniae resistance to complement-mediated killing were investigated by using different strains and isogenic mutants previously characterized for their surface components. We found that strains from serotypes whose K antigen masks the lipopolysaccharide (LPS) molecules (such as serotypes Ki, K1O, and K16) fail to activate complement, while strains with smooth LPS exposed at the cell surface (with or without K antigen) activate complement but are resistant to complementmediated killing. The reasons for this resistance are that C3b binds far from the cell membrane and that the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants (K+ or K-) are serum sensitive because they bind C3b close to the cell membrane and the lytic complex (C5b-9) is formed.

Both the classical and alternative complement pathways were effective in the elimination of K pneumoniae serumsensitive strains in nonimmune serum, although bacterial killing was more effective when both pathways were active. Activation of the classical pathway by these strains was studied in more detail, and we have identified two bacterial outer membrane proteins (OMP) that bind Clq and activate this pathway in nonimmune serum and in agammaglobulinemic (agamma) serum. MATERIALS AND METHODS Bacteria, bacteriophage, and media. The serum-sensitive K pneumoniae strain KT793 (0-:K-) used in this study is a double mutant obtained from the serum-resistant K pneumoniae strain C3 (serotypes O1:K66). It was made unencapsulated (K-) after UV mutagenesis and selection with anticapsular serum, as described before (5). Spontaneous LPSdeficient mutants (O-) were obtained from the Kmutant strain by selection with the LPS-specific bacteriophage FC3-2, as described previously (48). The other serum-sensitive strains used were KT701, KT702, and KT707, which were derived from the wild-type C3 strain and which were serotypes 0-:K66 (47). Strain KT707 shows only one OMP with an Mr of about 36,000 in the range of 35,000 to 40,000 (47). Strains KT717 (47) and KT791 (an isogenic mutant from strain C3) (5) are serum-resistant O1:Kstrains. Luria broth or nutrient broth was used for bacterial growth and phage propagation (33). Human sera. A pool of nonimmune human sera (NHS) was obtained from healthy volunteers. NHS diluted 1/100 did not react with OMP from K pneumoniae KT793 in Western blot (immunoblot) experiments. NHS was made deficient in Clq Vol. 61, No. 3

The role of lipopolysaccharide (LPS) in the susceptibility of Aeromonas hydrophila strains of serotype 0:34 to non-immune human serum was investigated using isogenic mutants (serum-sensitive), previously obtained on the basis of phage resistance, and characterized for their surface components. The classical complement pathway was found to be principally involved in the serum-killing of these sensitive strains. LPS preparations from serumresistant or serum-sensitive strains, or purified core oligosaccharides (low-molecular-mass LPS) inactivated both bactericidal and complement activity of whole serum, while the O-antigen molecules (high-molecular-mass LPS) did not. The results indicate that LPS core oligosaccharide composition contributes to complement resistance of A. hydrophila strains from serotype 0:34 with moderate virulence. IntroductionThe bactericidal effect of non-immune serum is important in immediate host defence against bacterial infections. This phenomenon has been widely studied (Morrison & Kline, 1977;Nelson & Roantree, 1967;Taylor, 1983Taylor, , 1988, and is known to be complementmediated (Roantree & Pappas, 1960). Although serum resistance in Gram-negative bacteria can be multifactorial in vivo, the outer membrane is predominantly important since it is usually the most peripheral component of the bacterial cell envelope. Outer membrane components such as lipopolysaccharide (LPS) (Munn et al., 1982;Mushel & Larsen, 1970;Nelson & Roantree, 1967), and proteins (Guyman et al., 1978;Hildebrant et al., 1978 ;Moll et al., 1980), as well as other components of the bacterial cell surface, such as capsular polysaccharides, have been implicated in resistance to the bactericidal activity of serum (Glynn & Howard, 1970;Rice & Kasper, 1977; Sutton et al., 1982).Complement activation by Gram-negative bacteria can occur via the classical complement pathway (CCP) or the alternative complement pathway (ACP). The CCP can be activated by the interaction of antibody with bacterial surface antigens or sometimes more directly by Abbreuiations: CCP, classical complement pathway; ACP, alternative complement pathway ; HMM-LPS, high-molecular-mass lipopolysaccharide; LMM-LPS, low-molecular-mass lipopolysaccharide; NHS, non-immune human serum ; KDO, 2-keto-3-deoxyoctulosonic acid (3-deoxy-D-rnanm-octulosonic acid).the lipid A moiety of LPS (Morrison & Kline, 1977). The ACP can normally be activated by bacterial polysaccharides independently of antibody (Quinn et al., 1977).Aeromonas hydrophila is both an opportunistic and a primary pathogen of a variety of aquatic and terrestrial animals, including humans (Howard & Buckley, 1985). The clinical manifestation of A. hydrophila infection ranges from gastroenteritis to soft tissue infections, septicaemia, and meningitis (Freij, 1984;Ljungh & Wadstrom, 1983). Surface characteristics, such as the presence of an S-layer or the type of LPS, permit classification of A . hydrophila into different categories on the basis of their virulence (Janda et al., 1985;Dooley et al., 1985). We r...

Growth ofAeromonas hydrophila strains from serotpe 0:34 at 20 and 37°C in tryptic soy broth resulted in changes in the lipids, lipopolysaccharide (LPS), and virulence of the strains tested. Cells grown at 20°C contained, relative to those cultured at 37rC, increased levels of the phospholipid fatty acids hexadecanoate and octadecanoate and reduced levels of the corresponding saturated fatty acids. Furthermore, the lipid A fatty acids also showed thermoadaptation. In addition, LPS extracted from cells cultivated at 20°C was smooth, while the LPS extracted from the same cells cultivated at 37C was rough. Finally, the strains were more virulent for fish and mice when they were grown at 20°C than when they were grown at 37C and also showed increased different extracellular activities when they were grown at 20°C. Aeromonas hydrophila is both an opportunistic and a primary pathogen of a variety of aquatic and terrestrial animals, including humans (31). The clinical manifestation of A. hydrophila infection ranges from gastroenteritis to softtissue infections, septicemia, and meningitis (11). Surface characteristics, such as the presence of an S-layer or the type of lipopolysaccharide (LPS), permit classification ofA. hydrophila into different categories on the basis of their virulence (9, 14). We recently described a group of A. hydrophila strains belonging to serotype 0:34 with heterogeneous O-polysaccharide chains in their LPS and without an S-layer, previously reported to be moderate in their virulence for fish (19, 23) and mice (23). Also, serotype 0:34 has been recently reported as one of the most frequently encountered among mesophilic Aeromonas species (35). Since A. hydrophila is ubiquitous in nature and also a pathogen, we are interested in the changes that temperature may have on its cellular chemistry, particularly as it affects membrane lipids, LPS, and outer membrane (OM) proteins, as well as their relationship with virulence. Studies on the effect of growth temperature on LPS chemistry have been largely restricted to members of the family Enterobacteriaceae and the genus Pseudomonas. Modifications of the composition of the lipid A component of LPS isolated from Salmonella species (41), Proteus mirabilis (34), Escherichia coli (38), and Yersinia enterocolitica (39) have been noted. Also, changes in the LPS have been observed at high temperatures on enterobacteria (20, 41) and Pseudomonas cells (16, 17), with the cells being smooth (S-form LPS) at low temperatures and rough (R-form LPS) at high temperatures. * Corresponding author. MATERIALS AND METHODS Bacteria, bactexiophages, and media. The strains used are listed in Table 1. Strains AH-3 and Ba5 were isolated from moribund fish, and strains AH-101, AH-102, and AH-103 were human clinical isolates. Bacteriophages PM1, PM2, and 18 from A. hydrophila were previously described by us (24, 25, 27). A. hydrophila strains were usually cultured on tryptic soy broth (TSB). TSB-agar was obtained by adding 1.5% agar and 0.6% TSB soft agar. Phage adsorption and sensi...

We obtained, by different methods, isogenic lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen) mutants from Klebsiella pneumoniae strains able to induce experimental infections (cystitis and pyelonephritis) in rats. We compared the induction of experimental infections in rats by wild-type strains and the lipopolysaccharide and capsular polysaccharide mutants. The high-molecular mass lipopolysaccharide of K. pneumoniae is clearly implicated in the infection process of the rat urinary tract, whilst the capsular polysaccharide seems not to be involved to the same extent.