Alginate‐producing, mucoid P. aeruginosa is frequently found in the lungs of patients with cystic fibrosis (CF), where it causes a chronic infection. The importance of alginate in the pathogenesis was demonstrated by the ability to establish chronic P. aeruginosa lung infection in rats if P. aeruginosa entrapped in minute alginate‐beads were inoculated transtracheally. Alginate beads containing P. aeruginosa were formed by nebulizing a suspension of seaweed sodium‐alginate and P. aeruginosa into a calcium solution. The alginate bead method of establishing infection was compared to an agar‐bead method and proved to be quantitatively similar after 4 weeks. The ability of the two methods to induce formation of precipitins, IgA and IgG antibodies against P. aeruginosa antigens, including outer membrane proteins, flagella, exoenzymes and alginate, was assessed by crossed immunoelectrophoresis, enzyme‐linked immunosorbent assay and immunoblotting. The two methods of inducing infection were comparable and infected rats had significantly higher antibody response than rats inoculated with sterile beads. We suggest that the alginate bead model closely resembles the later stages of CF‐lung infection and that it offers the theoretical advantage of using a substance which is chemically similar to the alginate produced in vivo by P. aeruginosa.
The development of significant mechanisms of resistance to beta-lactam antibiotics in Pseudomonas aeruginosa in cystic fibrosis (CF) patients have been studied in ten CF patients during a two week course of anti-pseudomonal beta-lactam antibiotic therapy. Sputum samples were collected on days 1, 7 and 15. Entire homogenized sputum samples were examined directly for the number of bacteria resistant to different levels of antibiotics. This allowed the detection of pre-existing resistant subpopulations of bacteria as well as following the changes in beta-lactam antibiotic susceptibility during treatment. P. aeruginosa isolates were characterized by means of sero-grouping, phage- and pyocin-typing. Outer membrane proteins of paired sensitive and resistant strains were characterized. Sonicated extracts of cells were assayed for basal and induced beta-lactamase activity. Beta-lactamase activity was further characterized by isoelectric focusing and inhibition profiles. Our observations were in accordance with the hypothesis that the sensitive inducible population was overrun by the pre-existing resistant subpopulation, during treatment. The resistant in-vivo selected P. aeruginosa population exhibited stable partially derepression but the beta-lactamase inhibitor tazobactam restored beta-lactam antibiotic activity.
Pseudomonas aeruginosa isolates from patients with cystic fibrosis were purified by repeated ethanol precipitation, nuclease digestion, anion-exchange chromatography, dialysis, and lyophilization. Uronic acid constituted 72% of the dry weight when mannuronolactone was used as the internal standard in the carbazole-borate assay for uronic acids. The average degree of acetylation was 16%, and the ratio of mannuronic acid to guluronic acid was 4.7. No homopolymeric blocks of guluronic acid were found when analyzed by nuclear magnetic resonance spectroscopy. Contaminating proteins were denatured by heating, and during purification the content of protein relative to alginate fell from 566 to 0.9%. The content of lipopolysaccharide was 0.012%. No immunological or biological activity was attributable to the protein or lipopolysaccharide content as estimated by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and a neutrophil chemotaxis assay. Rabbits were hyperimmunized with P. aeruginosa alginates and alginate from the seaweed Laminaria hyperborea, and an ELISA that detected alginate-specific antibodies was developed. Antibodies to P. aeruginosa alginate were detected by ELISA in 1:4,000 dilutions of serum from patients with cystic fibrosis with chronic P. aeruginosa lung infection. The serological cross-reactions between serum from the nine patients with cystic fibrosis and the corresponding P. aeruginosa alginates were investigated and showed considerable heterogeneity. This finding indicates that P. aeruginosa alginate from more than one P. aeruginosa strain should be used in serological tests. There was no serological cross-reactivity between P. aeruginosa and Laminaria hyperborea alginate in either rabbits or patients with cystic fibrosis.
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