Polymyxin B nonapeptide, derived by cleavage of the fatty acyl diaminobutyric acid from polymyxin B, is considerably less toxic, lacks bactericidal activity, and retains its ability to render gram-negative bacteria susceptible to several antibiotics by permeabilizing their outer membranes. The peptide rendered all 53 polymyxin-susceptible strains tested more susceptible to novobiocin, lowering the MIC of novobiocin eightfold or more. The combination of polymyxin B nonapeptide with novobiocin or with erythromycin administered intraperitoneally in multiple doses synergistically protected mice infected with gram-negative bacteria. This combination may be clinically useful because of the apparent rarity of the acquisition of resistance.Resistance to hydrophobic antibiotics in gram-negative bacteria may be either due to a decrease in the penetration of the antibiotic through the outer membrane or due to specific mechanisms, such as gene mutation or acquisition of resistance genes. The distinction between the two types of resistance is important, because it allows the search for antibiotics to which the development of specific mechanisms of resistance are rare but to which the bacterial membrane is impermeable. Such active but nonpenetrating antibiotics may gain clinical use when combined with membrane-active agents that increase their penetration. Polycations at sublethal concentrations increase the permeability of the outer membranes of gramnegative bacteria and render these microorganisms susceptible to many hydrophobic antibiotics (15)(16)(17)(18). Among the various polycations tested, polymyxin B nonapeptide (PMBN), derived by cleaving the fatty acid moiety from polymyxin B, has been studied the most. PMBN lacks antibacterial activity but renders most polymyxin-susceptible gram-negative bacteria susceptible to hydrophobic antibiotics by disorganizing the bacterial outer membranes to enhance antibiotic penetration (17)(18)(19). In the present study, we sought to further determine the incidence of strains that exhibit permeability-associated resistance in assays with PMBN and to examine the therapeutic efficacy of hydrophobic antibiotics in combination with PMBN in experimental gram-negative infections in mice.All clinical isolates were obtained from various body sites at regional outpatient clinics (see Table 2). The bacteria were grown on agar slants (nutrient agar; Difco Laboratories, Detroit, Mich.) and kept at 4°C for up to 2 weeks. The MICs of each antibiotic were determined as described elsewhere (7). 0.5 ml of phosphate-buffered saline (0.1 M NaCl, 0.02 M P04; pH 7.2) containing either 5 x 105 CFU of Klebsiella pneumoniae serotype K2 (1) harvested from the logarithmic phase of growth in nutrient broth (Difco) at 37°C or 1 x 108 CFU of Pseudomonas aeruginosa 33347 (kindly provided by Nehama Garber, Bar-Ilan University, Ramat-Gan, Israel) harvested from agar slants after overnight growth at 37°C (8).The efficacy of PMBN either alone or in combination with other antibiotics was evaluated following intraperiton...

We found that surfactant protein A (SP-A) enhances phagocytosis of Klebsiella pneumoniae K21a but not of K2 serotypes by alveolar macrophages. SP-A interacted with the capsule of K21a (containing Man alpha1 Man sequences) as shown by SP-A-induced agglutination of the bacteria, by binding of SP-A-coated particles onto the bacterial surface, and by binding of SP-A to immobilized parent K21a strain and recombinant strains that switched their capsule from K2 to K21a. In contrast, only marginal binding of SP-A to K2 parent strain (lacking this sequence) could be detected. Furthermore, binding of capsular polysaccharide of K21a to immobilized SP-A was inhibited by mannan but not by lipopolysaccharide and K2 capsular polysaccharide. SP-A-treated macrophages bound increased numbers of parent K21a strain and recombinant strains of K21a capsule type but considerably less parent K2 strain. SP-A also enhanced killing of K21a strains by macrophages. The enhanced binding of K21a by macrophages pretreated with SP-A was inhibited by mannan, suggesting that binding is mediated by the mannose receptor on macrophages. We conclude that SP-A increases phagocytosis of the Klebsiella by two mechanisms, one of which is by serving as an opsonin, which binds to the capsular polysaccharides of the bacteria and potentially to SP-A receptors on the macrophages, and the other by activating the macrophages, resulting in increased activity of the mannose receptor.

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of KiebsieUla pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK2la), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-ca-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumonwae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by-50 nm. Within this zone of separation, numerous and regularly spaced filaments of CPS were * Corresponding author. isolated. This analysis showed that the genes (cps) encoding CPS map close to his and are probably clustered. Recently, the genes determining K pneumoniae K2 CPS were cloned as a cps gene cluster occupying more than 15 kb of ...

Klebsiella pneumoniae strains of the K2 capsular serotype are usually highly virulent in mice, which is in contrast to the low virulence of most other serotypes. Here we used a genetic approach to examine the relative contribution of capsule type to the virulence of K. pneumoniae in mice. We used wild-type strains expressing capsular polysaccharide (CPS) serotypes K2 (strain KPA1) and K21a (strains KPB1 and KPC1), which were then used to construct capsule-switched derivatives. The close proximity of the cps gene cluster to selectable his markers made it possible to mobilize the cps genes by conjugation from one serotype (donor) to another (recipient) and to obtain recombinants in which interserotype switching had occurred by reciprocal recombination. Each capsule-switched derivative examined of the KPA and KPC strain backgrounds produced a CPS that was immunologically and structurally identical to that of the donor. Strain background was confirmed by demonstrating restriction fragment length polymorphism patterns identical to those of the respective recipients. The parent strains were then compared with capsule-switched recombinants for phenotypic properties associated with virulence. Clearance from the bloodstreams of mice was rapid in serotype K21a strains of either wild-type or recombinant origin, whereas K2 strains remained viable in the blood during the period examined. These differences appeared to be dependent upon the CPS type but independent of strain background. Binding to macrophages was higher in K21a strains than in those with the K2 capsule and was also independent of the strain background. Both blood clearance and macrophage-binding activities were completely inhibited by yeast mannan, suggesting that they were mediated via the macrophage mannose receptor. The K2 parent strain was highly virulent to mice (50% lethal dose [LD 50 ], 3 ؋ 10 3), while the K21a parent strains demonstrated low virulence (LD 50 , >2 ؋ 10 8). Interestingly, the virulence of recombinant KPC10(cpsK2), originally of the KPC1(cpsK21a) background, was intermediate (LD 50 , 4 ؋ 10 5). In contrast, both cpsK21a recombinants of the originally virulent KPA1(cpsK2) background became nearly avirulent (LD 50 , >2 ؋ 10 8). Six additional serotypes (K12, K24, K32, K55, K62, and K67) were examined, and all showed a positive correlation between the ability of the Klebsiella serotype to interact with a human mannose receptor, as expressed by Cos I cell recombinants, and the LD 50 of the serotype. These results suggest that expression of a capsule which is recognized by the mannose receptor markedly affects the interaction with macrophages and blood clearance. The virulence of the cpsK2 recombinant of the KPC background may have been enhanced because it was expressing a heterologous capsule not recognized by the mannose receptor. Thus, this study shows that the capsule type plays an important role in the rate of blood clearance and phagocytosis but contributes only partially to the virulence of K. pneumoniae in mice.

Recognition and phagocytosis of micro-organisms in a serum-poor environment represent innate immunity against many extracellular pathogens. As a paradigm for such processes, we discuss the recognition of Klebsiella pneumoniae by alveolar macrophages and monocyte-derived macrophages in the absence of serum. Macrophages recognize and subsequently kill Klebsiella expressing Man-alpha 2/3-Man or Rha-alpha 2/3-Rha sequences in their capsular polysaccharides by two mechanisms: (a) recognition of the capsular structures by macrophage mannose receptors, and (b) opsonization by the lung surfactant protein A (SP-A), which binds to the capsular polysaccharides of Klebsiella and to SP-A receptors on the macrophages. Sp-A may also enhance phagocytosis by increasing the activity of macrophage mannose receptors. We conclude that a specific microbial surface structure may be a target for recognition by macrophages via several mechanisms, as exemplified in the case of Klebsiella capsular polysaccharides. Multiple recognition mechanisms of pathogens by macrophages may be essential to provide innate immunity to reduce the frequency of infections caused by a relatively less virulent bacterium in the immuno-compromised host.