Polysaccharide surface antigens expressed by nonmucoid isolates of Pseudomonas aeruginosa from cystic fibrosis patients.

We tested nonmucoid Pseudomonas aeruginosa isolates obtained from cystic fibrosis (CF) patients for the expression of lipopolysaccharide (LPS) serotype antigens, serum sensitivity, and production of mucoid exopolysaccharide (MEP). When all nonmucoid isolates were compared with a set of random mucoid isolates, 20 of 52 (38%) nonmucoid isolates were typable and serum resistant, compared with 13 of 51 (24%) mucoid isolates (P = 0.16 by chi-square analysis). However, nonmucoid strains from CF patients colonized only with nonmucoid strains were more frequently typable and serum resistant (67%) than were nonmucoid isolates from patients cocolonized with mucoid strains (31%) (P = 0.012, Fisher exact test). An inhibition enzyme-linked immunosorbent assay done with bacterial extracts, a direct-whole-cell enzyme-linked immunosorbent assay done with affinity-purified antibody to MEP, and immune electron microscopy all demonstrated production of MEP by all nonmucoid P. aeruginosa isolates tested, including nonmucoid revertants of mucoid strains. No other bacterial species tested positive in these assays. These findings suggest that MEP is produced by all P. aeruginosa isolates obtained from CF patients, that the initial colonizing nonmucoid strains produce a smooth LPS, and that once LPS-rough, mucoid strains appear in the sputum, the predominant LPS phenotype is rough regardless of colony morphology.     

Serotyping of Pseudomonas aeruginosa isolates from patients with cystic fibrosis of the pancreas.

Pseudomonas aeruginosa isolates (173) from 144 patients with cystic fibrosis (CF) of the pancreas in seven hospitals were serotyped with the agglutination systems of Homma (1974) and Fisher et al. (1969). The two systems were complementary. Strains from CF patients were much less likely to furnish a stable type on repetitive typing tests than strains from other patients. This was related to the frequent occurrence of mucoid P. aeruginosa strains. The 173 strains were divided among 11 Homma serotypes. A single Homma type (type 8) capable of mucoid growth comprised 104 (60%) CF strains. Eight serotypes were detected in 77 strains from 48 CF patients in one hospital; three strains were detected in one hospital CF unit; and two strains were detected in each of five hospital CF units. The CF serotype comprised from 50 to 93% of CF strians inthe seven hospitals. These P. aeruginosa strains dissociated in vivo as judged by mucoid and nonmucoid colonies on primary culture plates and continued to dissociate during subcultures. Both colony type were the same serotype. The tendency to regard colonial phenotypes (mucoid, nonmucoid, rough) as separate strians was erroneous. Repetitive typing with the two systems gave better results than a single system. The mucoid P. aeruginosa strain is probably spread from patient to patient, rather than acquiring its mucoid characteristic de novo in the CF patient. It is not known why the mucoid CF strain has a peculiar predilection for CF patients, nor why it generally loses the quality in culture but retains it indefinitely in the patient.     

Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis.

Isolates of Pseudomonas aeruginosa from cystic fibrosis patients are unusual; they are often susceptible to the bactericidal effect of human serum, have a rough lipopolysaccharide, and produce an exopolysaccharide that is responsible for the characteristic mucoid phenotype. In contrast, strains from the environment and from patients with other diseases usually have smooth lipopolysaccharide, do not produce very much mucoid exopolysaccharide, and are phenotypically nonmucoid. The predominance of mucoid strains of P. aeruginosa in infections of patients with cystic fibrosis has not been explained. In the lower airways, where P. aeruginosa persists in cystic fibrosis, nutrients for bacterial growth may be limited. We investigated whether growth of P. aeruginosa under conditions of suboptimal nutrition causes conversion to the characteristic cystic fibrosis phenotype. Ninety-two strains of P. aeruginosa were maintained for up to 90 days in a minimal medium with acetamide as the sole carbon source. In 56 (52%) of 107 cultures, isolates with rough lipopolysaccharide emerged, and in 20 (19%) of 104 nonmucoid cultures, mucoid isolates were recovered. Strains with rough lipopolysaccharide also were sensitive to the bactericidal effect of normal human serum. Under conditions of suboptimal nutrition in vitro, isolates of P. aeruginosa emerged that produced rough lipopolysaccharide and were mucoid, typical of many isolates from cystic fibrosis patients. This peculiar phenotype may arise as a consequence of nutritional limitation within the cystic fibrosis respiratory tract rather than from features unique to these strains of bacteria.     

Mucoid Escherichia coli in cystic fibrosis

Patients with cystic fibrosis commonly harbor in their lungs strains of Pseudomonas aeruginosa that have a mucoid coating considered virtually pathognomonic for the disease. We found that strains of Escherichia coli with a morphologically similar mucoid coating were present in the respiratory tracts of eight (11.8 per cent) of 68 patients with cystic fibrosis whose sputum cultures yielded Esch. coli, as compared with none of 89 patients without cystic fibrosis who had Esch. coli in sputum. Mucoid strains of Esch. coli were also recovered from the stools of five (11.1 per cent) of 45 patients with cystic fibrosis, as compared with one (0.7 per cent) of 150 patients without cystic fibrosis. The mucoid substances purified from Esch. coli were biochemically and antigenically distinct from those of P. aeruginosa. We conclude that the respiratory tract in cystic fibrosis offers an environment conducive to the production of a mucoid coating not only by P. aeruginosa but by other gram-negative bacilli as well.     

Transfer of a chromosomal locus responsible for mucoid colony morphology in Pseudomonas aeruginosa isolated from cystic fibrosis patients to P. aeruginosa PAO

The locus responsible for mucoid colony morphology in five independent clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients have been transferred by means of pM060-mediated conjugation to the genetically characterised strain P. aeruginosa PAO. Genetic mapping has shown that in all five strains the locus is on the chromosome between 89' and 94', although it is not possible to say that the same locus is involved in each case. The way is now open for a more detailed genetic analysis of the loci responsible for mucoid colony morphology.     

Marked phenotypic variability in Pseudomonas cepacia isolated from a patient with cystic fibrosis.

Characterization of the epidemiology of Pseudomonas cepacia colonization in cystic fibrosis is difficult because of the phenotypic variability of isolates. A single sputum culture may yield colonies which differ in morphology, antibiotic susceptibility, and pigment production. We examined serial P. cepacia isolates from a cystic fibrosis patient which the clinical laboratory identified as separate strains; these were selected on the basis of isolation date and culture site. An attempt was made to sample at multiple time points and, at a single time point, from three different culture sites. Ribotype analysis, using both the standard Southern blot technique and a recently reported method which uses the polymerase chain reaction, was used to distinguish unique P. cepacia strains. Characterization included comparison of antibiotic susceptibility, plasmid content, and outer membrane protein (OMP) patterns. rRNA analysis demonstrated that all isolates had the same ribotype, consistent with their being derivatives of the same strain. Antibiotic susceptibility testing revealed variability among both same-date and same-site isolates. Screening for plasmid DNA identified three groups of isolates; both same-date and same-site isolates demonstrated variability. OMP profiles were similar, but at least six distinct patterns were identified. For the six same-date isolates, five different OMP patterns were identified. For the 10 same-site isolates from different dates, five of the six OMP patterns were represented. We have demonstrated marked phenotypic variability in 14 strains of P. cepacia isolated from different sites and at different times from a single colonized patient. Ribotyping identified all the isolates as derivatives of a single strain; thus, the diversity of phenotypes appears to be the result of differential gene expression.     

Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis.

Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for pulmonary deterioration of cystic fibrosis patients. The purpose of this study was to type the P. aeruginosa isolates collected sequentially from cystic fibrosis patients, chronically colonized with P. aeruginosa, by random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR). Sequential P. aeruginosa isolates (n: 130) that had been collected from 20 CF patients over at least 9 years were investigated. The isolates were analyzed by RAPD-PCR using two arbitrary primers. Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method. RAPD-PCR typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype. Some CF patients were colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype. However, some patients may be colonized with more than one genotype. The results also demonstrated that there might be a risk of cross-colonization between CF patients followed-up at the same center.     

Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis.

Pseudomonas aeruginosa isolates recovered from chronically colonized patients with cystic fibrosis (CF) are phenotypically different from those collected from other patients or from the environment. To assess whether alterations in motility, mucoidy, and serum susceptibility represented an adaptation to chronic infection or replacement by a new strain, sequential P. aeruginosa isolates of known phenotype collected from 20 CF patients were typed by random amplified polymorphic DNA (RAPD) analysis. A total of 35 RAPD strain types were found among 385 isolates from 20 patients, and only two patients had P. aeruginosa strains of the same RAPD fingerprint. Eight strain pairs representative of the first eight RAPD types were also analyzed by SpeI macrorestriction followed by pulsed-field gel electrophoresis (PFGE); the strain types found by both fingerprinting techniques correlated exactly. In 11 of 20 patients, the RAPD types of serial P. aeruginosa isolates remained stable despite alterations in isolate motility, colonial morphology, and lipopolysaccharide phenotype. However, in isolates collected from one CF patient, a single band change in RAPD fingerprint and CeuI PFGE profile correlated with the appearance of an RpoN mutant phenotype, suggesting that the altered phenotype may have been due to a stable genomic rearrangement. Secretion of mucoid exopolysaccharide, loss of expression of RpoN-dependent surface factors, and acquisition of a serum-susceptible phenotype in P. aeruginosa appear to evolve during chronic colonization in CF patients from specific adaptation to infection rather than from acquisition of new bacterial strains.     

Phenotypic conversion of Pseudomonas aeruginosa in cystic fibrosis.

Pseudomonas aeruginosa strains isolated from cystic fibrosis patients were tested for production of exoenzymes, sensitivity to pooled normal human serum, and colony morphology. Strains isolated from patients exhibiting a severe form of the disease were seen to produce a decreased range of exoenzymes, to show an increase in their serum sensitivity, and to be predominantly mucoid in colonial character compared with strains isolated from patients with a milder form of the disease. These results suggest that P. aeruginosa undergoes phenotypic changes with respect to exoenzyme secretion, serum sensitivity, and colony form as the clinical condition of the cystic fibrosis patient changes.     

Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa.

Laboratory-derived mucoid variants of Pseudomonas aeruginosa were selected by plating the standard PAO1 laboratory strain with bacteriophage. These mucoid variants formed two distinct groups of strains on the basis of phage typing. The first group had the same phage-typing pattern as the parent PAO1 strain, while the second group had a distinctly different phage-typing pattern. One strain from each group was assessed along with the parent PAO1 strain for its outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles by sodium dodecyl sulfate-gel electrophoresis followed by appropriate staining. The mucoid derivatives were found to differ from the parent PAO1 nonmucoid strain in having lost a high-molecular-weight LPS species. Furthermore, the reversion of the mucoid strains to the nonmucoid phenotype was accompanied by a return of the missing high-molecular-weight LPS species. No observable difference between the mucoid derivatives and the parent nonmucoid strain was noted in the OMP profiles. The opposite was found in the case of four isolates of mucoid P. aeruginosa from patients with cystic fibrosis. Two OMP bands (of approximately 55 and 25 kilodaltons) were present in the mucoid isolates but missing in their sister nonmucoid strains. In the case of the cystic fibrosis isolates, no difference in the LPS profiles within mucoid-nonmucoid pairs was noted.     

Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis.

The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis.     

Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms

Once mucoid (alginate-producing) strains of Pseudomonas aeruginosa have become established in the respiratory tracts of cystic fibrosis patients they can rarely be eliminated by antibiotic treatment alone; we have investigated, in an in vitro biofilm system, the putative role of co-administration of alginate lyase with antibiotic. Biofilms were maintained in continuous flow culture in a medium resembling sputum from CF patients. Antibiotics and/or alginate lyase were added to some of the cultures. Biofilms of two mucoid CF strains of P. aeruginosa were, in most cases, not eradicated by a one-week course of treatment with 64 microg/ml of gentamicin; the same concentration of gentamicin, under the same conditions, led to the apparent elimination of all biofilms of non-mucoid derivatives of these strains. When alginate lyase and gentamicin were administered together the apparent elimination of mucoid bacteria from biofilms was achieved, whereas the mucoid bacteria in most control biofilms treated only with gentamicin persisted. Ceftazidime treatment of biofilms was more effective against those containing the non-mucoid strains than those with mucoid strains. These studies support the view that co-administration of antibiotics with alginate lyase, which degrades the exopolysaccharide produced by mucoid strains of P aeruginosa, might benefit CF patients by increasing the efficacy of antibiotic in the respiratory tract.     

Phenotypic differences among clinically isolated mucoid Pseudomonas aeruginosa strains

Mucoid strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis or urinary tract infections displayed many phenotypic differences. The ratios of D-mannuronosyl to L-guluronosyl moieties of the extracellular alginate-like polysaccharides produced by the 19 strains examined varied from 99 to 0.8; the acetyl content of the polymers varied from 0.38 to 0.02 mol per mole of uronosyl residue. The strains also differed with regard to the stability of the mucoid phenotype. Of 15 isolates from patients with cystic fibrosis, 7 displayed stable mucoid phenotypes; 8 isolates were unstable and reverted to the nonmucoid phenotype at high frequency. The four strains isolated from patients with urinary tract infections were also unstable. Strains from urinary tract infections expressed the mucoid phenotype on six different media, both minimal and complex, whereas cystic fibrosis-associated strains varied widely with regard to medium-dependent expression of the mucoid phenotype. Of 15 cystic fibrosis strains, 5 were mucoid on each of six different media, 4 were mucoid on five media, 1 was mucoid on four media, 4 were mucoid on three media, and 1 yielded mucoid colonies on only one of the six media tested. There was no obvious correlation among polysaccharide structure, stability of the mucoid phenotype, and medium-dependent expression of the mucoid phenotype for any of the 19 strains investigated. These data suggest that although mucoid strains of P. aeruginosa must share some common property related to their ability to colonize their host, this property seems to be unrelated to polysaccharide composition, medium-dependent expression of the mucoid phenotype, or stability of the mucoid phenotype.     

Pseudomonas aeruginosa: changes in antibiotic susceptibility, enzymatic activity, and antigenicity among colonial morphotypes

Colonial variants of Pseudomonas aeruginosa have received renewed interest because of their occurrence in sputum cultures of patients with cystic fibrosis. We encountered 11 strains of P. aeruginosa from various body sites of non-cystic fibrosis patients. The strains showed two to three colonial variants, including smooth, rough, and iridescent morphotypes that arose from subculture of a single colony of P. aeruginosa originating from a primary source. The colonial segregants differed in antibiotic susceptibility (resistance to gentamicin, carbenicillin, chloramphenicol, and tetracycline), presence or absence of exoenzymes (gelatinase and elastase), degree of proteolytic activity (caseinase), pigmentation, and antigenicity. These observations suggest that in vivo dissociation with concomitant changes in enzymatic and surface properties might greatly enhance invasiveness. Concurrent differences in antimicrobial susceptibility among the colonial variants could account in some instances for the failure of antibiotic treatment in P. aeruginosa infections in which one would anticipate a positive therapeutic response.     

Alginase treatment of mucoid Pseudomonas aeruginosa enhances phagocytosis by human monocyte-derived macrophages.

Mucoid Pseudomonas aeruginosa colonizes and infects the respiratory tract of most older patients with cystic fibrosis. These bacteria resist both opsonin-dependent and -independent phagocytosis by human polymorphonuclear leukocytes and monocyte-derived macrophages. Resistance to phagocytosis is thought to be mediated in part by the mucoid exopolysaccharide associated with the bacterial surface. The purpose of this study was to determine whether degradation of the mucoid exopolysaccharide by alginase enhances bacterial susceptibility to nonopsonic phagocytosis by macrophages. Eight phagocytosis-resistant mucoid P. aeruginosa isolates from patients with cystic fibrosis were studied. The bacteria were treated with a preparation of alginase from Bacillus circulans, and phagocytosis by macrophages was measured by a visual inspection assay. Alginase degradation of mucoid exopolysaccharide was measured by the periodate-thiobarbituric acid assay and by indirect immunofluorescence with a mouse monoclonal antibody to the mucoid exopolysaccharide. Alginase degraded the mucoid exopolysaccharide of all eight mucoid strains tested. Phagocytosis was enhanced in five of the eight strains. Alginase-enhanced phagocytosis was magnesium dependent and heat labile. Alginase may be a useful tool for studying the biological properties of P. aeruginosa mucoid exopolysaccharide.     

Accuracy and cost of antibiotic susceptibility testing of mixed morphotypes of Pseudomonas aeruginosa.

Chronic Pseudomonas aeruginosa colonization of the lower respiratory tract of patients with cystic fibrosis frequently results in pulmonary exacerbations requiring treatment with antimicrobial agents. Multiple morphotypes with different antibiotic susceptibilities are often isolated from a single sputum sample. Determination of MICs of antibiotics for each sputum morphotype is used to guide therapy but is time-consuming and expensive. We explored an alternative assay for determining MICs for all P. aeruginosa morphotypes cultured from a homogenized sputum sample. We sought correlations of those MICs with the MIC for the most resistant morphotypes tested separately. The MICs determined for a mixture of morphotypes correctly predicted the highest MICs (+/- one dilution) determined for isolated morphotypes 73.5% of the time. The MIC for the mixed morphotypes correctly predicted susceptibility in 90.4% of samples. In contrast, determination of the MIC for the mixture of morphotypes correctly predicted resistance in only 57.0%. For sputa containing susceptible isolates, testing the mixed culture may provide adequate susceptibility data with significant laboratory time and cost savings. However, for sputa with resistant strains, the traditional method of testing isolated morphotypes should still be used.     

Single and combination antibiotic susceptibilities of planktonic,adherent, and biofilm-grown Pseudomonas aeruginosa isolates cultured from sputa of adults with cystic fibrosis

Evidence suggests that Pseudomonas aeruginosa bacteria form biofilms within the airways of adults with cystic fibrosis (CF). The objective of this study was to determine whether clinical isolates of P. aeruginosa recovered from adults with CF have similar susceptibilities to individual antibiotics and to antibiotic combinations when grown as adherent monolayers or as biofilms compared to when they are grown using planktonic methods. Twelve multiresistant P. aeruginosa isolates, one mucoid and one nonmucoid from each of six CF patients, were grown conventionally under planktonic conditions, as adherent bacterial monolayers, and as biofilms. Each bacterial isolate remained genotypically identical despite being cultured under planktonic, adherent, or biofilm growth conditions. Isolates grown as adherent monolayers and as biofilms were less susceptible to bactericidal killing by individual antibiotics compared to those grown planktonically. More importantly, biofilm-grown bacteria, but not adherent monolayer-grown bacteria, were significantly less susceptible to two- and three-drug combinations of antibiotics than were planktonically grown bacteria (P = 0.005). We conclude that biofilm-grown bacteria derived from patients with CF show decreased susceptibility to the bactericidal effects of antibiotic combinations than do adherent and planktonically grown bacteria.     

Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin.

Growth and exoproduct production were examined with sputum from patients with respiratory diseases serving as the growth substrate for mucoid strains of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients. Mucoid strains are uniquely common to chronic respiratory infections of CF patients. The mucoid colonial morphology of P. aeruginosa is due to the biosynthesis of the exopolysaccharide alginate. Alginate-producing (Alg+) strains utilized CF sputum for growth and high yields of alginate; however, sputum from patients with other respiratory diseases produced comparable results. Analysis of CF sputum medium indicated that amino acids and small peptides were major substrates for P. aeruginosa in respiratory secretions. Cultures of Alg+ strains in CF sputum medium were inhibited in growth and reduced in alginate yields by a low concentration (1 mM) of D-mannose, suggesting therapeutic applications. The rates of growth of two Alg+ strains in CF sputum medium were found to be slightly lower compared with their respective spontaneous Alg- mutants, indicating that the mucoid phenotype does not enhance the ability of P. aeruginosa to utilize respiratory secretions. At all stages of growth in CF sputum medium, two Alg+ strains produced lower yields of protease than did their respective Alg- mutants. When seven Alg+ strains of CF origin were compared with their respective Alg- mutants, the Alg+ phenotype correlated with reduced yields of extracellular proteases. These data are consistent with the hypothesis that mucoid strains of P. aeruginosa are more suited to chronic rather than to acute respiratory infections in that reduced yields of proteases temper the level of damage to the lungs and result in a reduced infiltration of phagocytic cells.     

Comparison of agar diffusion methodologies for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of patients with cystic fibrosis (CF). Improved antimicrobial chemotherapy has significantly increased the life expectancy of these patients. However, accurate susceptibility testing of P. aeruginosa isolates from CF sputum may be difficult because the organisms are often mucoid and slow growing. This study of 597 CF isolates of P. aeruginosa examined the correlation of disk diffusion and Etest (AB BIODISK, Solna, Sweden) results with a reference broth microdilution method. The rates of interpretive errors for 12 commonly used antipseudomonal antimicrobials were determined. The disk diffusion method correlated well (zone diameter versus MIC) for all of the agents tested. However, for mucoid isolates, correlation coefficients (r values) for piperacillin, piperacillin-tazobactam, and meropenem were <0.80. The Etest correlation with reference broth microdilution results (MIC versus MIC) was acceptable for all of the agents tested, for both mucoid and nonmucoid isolates. Category interpretation errors were similar for the disk diffusion and Etest methods with 0.4 and 0.1%, respectively, very major errors (false susceptibility) and 1.1 and 2.2% major errors (false resistance). Overall, both agar diffusion methods appear to be broadly acceptable for routine clinical use in susceptibility testing of CF isolates of P. aeruginosa.     

Isolation of mucoid strains of Pseudomonas aeruginosa from non-cystic-fibrosis patients and characterisation of the structure of their secreted alginate

When the incubation period of primary isolation plates was extended to 48 h, mucoid strains of Pseudomonas aeruginosa were found in specimens from various infected sites in patients who did not have cystic fibrosis. The 17 mucoid isolates were characterised in terms of mucoid type, pyocin type, and their sensitivity or resistance to seven beta-lactam and two aminoglycoside antibiotics. The carbohydrate, uronic acid (alginate) and protein content of the water-soluble extracellular material of 15 strains was determined. This material was fractionated by ion-exchange chromatography, and the presence of alginate confirmed by the chemical assay of uronic acids and their quantitation by gas-liquid chromatography. Uronic acids were absent from a non-mucoid revertant of one strain. The strains produced alginate with a high content of mannuronic acid and substituted with O-acetyl groups. By proton nuclear magnetic resonance (1H-nmr) analysis the alginate from three strains was shown to lack polyguluronate blocks in its structure. These properties are also found in the alginate of mucoid P. aeruginosa strains from patients with cystic fibrosis.