Influence of nutrient media on the chemical composition of the exopolysaccharide from mucoid and non-mucoid Pseudomonas aeruginosa

Two mucoid Pseudomonas aeruginosa strains and their non-mucoid revertants isolated from two different clinical origins (cystic fibrosis and bronchiectasis) were grown in various chemically defined media. The extracted exopolysaccharide was characterized by gas-liquid chromatography and 1H-NMR spectroscopy. The exopolysaccharide was always heterogeneous, with an alginate fraction and a neutral fraction essentially composed of glucose, galactose, rhamnose and hexosamines. The alginate composition (mannuronate/guluronate ratio and O-acetylation degree) changed according to the carbon source in nutrient media and whether the strains tested were responding differently to these environmental stimuli. In all cases, the best carbon source for the alginate production was glycerol: the two cystic fibrosis strains produced a predominantly O-acetylated alginate whereas only the mucoid bronchiectasis strain produced a polymannuronate exopolysaccharide.     

Comparative ultrastructure of a mucoid strain of Pseudomonas aeruginosa isolated from cystic fibrosis patient and its spontaneous non-mucoid mutant

The ultrastructure of a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin and its spontaneous non-mucoid variant was compared by transmission electron microscopy. Negatively-stained preparations of the mucoid strain obtained from plate cultures demonstrated dense, fibrous material projecting from the cell. No such material was observed in thin-sections or in negatively-strained preparations from liquid cultures. Thin-sections of ethanol-precipitated extracellular material from liquid cultures of the mucoid-strain revealed a cottony mesh of thin electron dense fibres. The non-mucoid strain did not produce such material. When prefixed with glutaraldehyde/malachite green mixture, cells of both strains demonstrated electron dense intracellular and extracellular malachite green-stainable structures. The internal complexes were frequently associated with the nucleoid or cell membrane and were replaced by electron transparent areas in cells prefixed with glutaraldehyde alone. Aeruginocins of the R-type were observed in mitomycin C induced cultures of both strains. Bacteriophages with 'claw-shaped' tail-tips were observed in the mucoid strain. Crystalline material was produced by the mucoid strain but only when plated on certain media.     

The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa

Cells of mucoid and non-mucoid Pseudomonas aeruginosa in colonies were at least one-thousandfold less sensitive to the antibiotics tobramycin or cefsulodin than were cells of the same bacteria in dispersed suspension. We did not detect any difference between the mucoid form and the non-mucoid form in the antibiotic sensitivity of colonies, from which we infer that the exopolysaccharide of the mucoid form does not contribute to colony-resistance by forming a barrier to antibiotic diffusion. Mathematical models were constructed in order to estimate time-courses of penetration of tobramycin and cefsulodin into biofilms and microcolonies of mucoid and non-mucoid P. aeruginosa. For tobramycin penetration, adsorption of antibiotic to the exopolysaccharide of the glycocalyx and antibiotic uptake by cells were taken into account in the calculations. The longest time-period for the concentration of tobramycin at the base of a biofilm 100 micron deep to rise to 90% of the concentration outside the biofilm was predicted to be 2.4 h. For cefsulodin penetration, irreversible hydrolysis catalysed by beta-lactamase was taken into account, using beta-lactamase levels taken from the literature. The calculations predicted that the cefsulodin concentration at the base of a biofilm 100 micron deep would rise to 90% of the external concentration in 29 s when the beta-lactamase was synthesized at the basal level. For a similar biofilm of bacteria synthesizing enhanced levels of beta-lactamase ('derepressed'), the concentration of cefsulodin at the base was calculated to rise to 41% of the external concentration in about 50 s and then remain at that level. This was despite the fact that cefsulodin is a poor substrate for this beta-lactamase.     

Comparative study of the biological characteristics of mucoid and non-mucoid Pseudomonas aeruginosa strains isolated from chronic respiratory infections

Morphological, culture and enzymatic characteristics, as well as virulence, susceptibility to antimicrobial agents and epidemiological markers, were studied for 100 mucoid and 100 non-mucoid Ps. aeruginosa strains isolated from chronic respiratory infections. For 10 mucoid and 10 non-mucoid strains was performed the active protection test in mice, both with inactivated germs (10(9) germs), and LPS extracted by Westphal method. It was ascertained that mucoid Ps. aeruginosa strains differ from non-mucoid strains by the slow growth on culture media, more reduced proteolytic activity (81% as compared to 99%), slow oxidation of carbohydrates (5-7 days), reduced virulence in mice (8%) or avirulence (92%), higher sensitivity to some antibiotics (amikacin, dibekacin, ticarcillin, tetracycline, cefotaxime, ceftriaxone), lysoresistance (74%) and polyagglutinability (67%). The mucoid strains ensure a reduced active protection in mice, 70% of strains did not protect the mice against the infection with virulent homologous strains, while the non-mucoid strains ensured 80%-100% protection.     

Exopolysaccharide production by mucoid and non-mucoid strains of Burkholderia cepacia

Thirteen strains of Burkholderia cepacia from various origins with mucoid and non-mucoid phenotypes were assayed for exopolysaccharide (EPS) production. The EPS were characterized by glycosyl composition analysis and examination of the products resulting from lithium-ethylenediamine and Smith degradations. The results showed that all strains, including the non-mucoid strains, were able to produce EPS exhibiting the same structural features, i.e. presence of one rhamnosyl, three galactosyl, one mannosyl, one glucosyl and one glucuronosyl residues, suggesting that this EPS is representative of the B. cepacia species.     

Identification of a slime exopolysaccharide depolymerase in mucoid strains ofPseudomonas aeruginosa

Strains ofPseudomonas aeruginosa recovered from pulmonary infections in cystic fibrosis (CF) patients are often mucoid in appearance owing to the secretion of a viscous slime exopolysaccharide (EPS). Unlike most mucoid isolates, strains WcM#2, P10, and P11 produce mucoid colonies after 24 h of incubation at 37°C, which become nonmucoid upon further incubation; this suggests the presence of a slime-degrading enzyme or depolymerase. Using both qualitative and quantitative assays, the presence of a slime EPS depolymerase was confirmed in each of these three strains as well as in four of four additional mucoid strains. Depolymerase activity was lower but still detectable in four of four nonmucoid strains. Enzyme preparations from strains WcM#2, P10, and P11 were active on most, but not all, slime EPS preparations fromP. aeruginosa strains, as well as sodium alginate; greater activity was observed on substrates after deacetylation. Comparisons are made between the enzyme described in this study and previous reports of slime EPS depolymerase in mucoid strains ofP. aeruginosa.     

Continuous culture studies on the synthesis of capsular polysaccharide by Klebsiella pneumoniae K1

The synthesis of capsular polysaccharide by Klebsiella pneumoniae K1 was investigated in a minimal salts medium by continuous culture. The organism produced larger amounts of polysaccharide under nitrogen-limited conditions than under carbon-limited conditions. The synthesis of polysaccharide was dependent not only on the availability of excess carbon, but also on growth rate. The rate of polysaccharide synthesis was greatest at low dilutions, low temperature (30°C) and at neutral pH. Prolonged growth in nitrogen-limited culture resulted in the development of non-mucoid variants, possibly due to a selective growth advantage over mucoid cells. The non-mucoid isolate was more susceptible to some bacteriophages, possibly due to the reduction or absence of capsular polysaccharide.     

Different mutations in mucA gene of Pseudomonas aeruginosa mucoid strains in cystic fibrosis patients and their effect on algU gene expression

Alginate biosynthesis in Pseudomonas aeruginosa is a highly regulated process in which algU and mucA genes are key elements. Mutations in mucA gene determine alginate operon overexpression and exopolysaccharide overproduction. In our study, 119 strains of P. aeruginosa were isolated from sputa of 96 cystic fibrosis patients and 84/119 showed nonmucoid phenotype, while 35/119 showed mucoid phenotypes. mucA gene was amplified and sequenced in all strains revealing mutations in 29/35 mucoid strains (82%) and in one non-mucoid strain. 4/29 strains showed mutations never described that generated premature stop and much shorter MucA proteins. In all mutated strains, algU gene expression was analyzed to determine if mutations in mucA, resulting in a strong loss of its protein, could significantly influence its function and subsequently the biosynthetic pathways under algU control. Analysis of algU expression disclosed that the length significantly affects the expression of genes involved in the production of alginate and in the motility and hence survival of P. aeruginosa strains in cystic fibrosis lungs.     

Partial purification and characterization of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis.

An exopolysaccharide depolymerase was isolated from a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin. Purified preparations of the depolymerase showed maximum activity against the unacetylated polymannuronic acid exopolysaccharide from the same strain and little activity against commercially prepared alginic acid. The evidence suggests that the enzyme is either periplasmic in location or associated with the outer cell membrane and is released extracellularly, in the absence of cell lysis, after a reduction of the culture magnesium (Mg2+) concentration below 3.0 mM. The depolymerase is also released after the addition of sublethal concentrations of EDTA to cultures containing 3.0 mM Mg2+. A survey of additional mucoid P. aeruginosa isolates recovered from patients with cystic fibrosis showed that nearly 60% demonstrated similar depolymerase activity while none of the nonmucoid revertants of the parent strains produced detectable depolymerase activity.     

Partial purification and characterization of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis

An exopolysaccharide depolymerase was isolated from a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin. Purified preparations of the depolymerase showed maximum activity against the unacetylated polymannuronic acid exopolysaccharide from the same strain and little activity against commercially prepared alginic acid. The evidence suggests that the enzyme is either periplasmic in location or associated with the outer cell membrane and is released extracellularly, in the absence of cell lysis, after a reduction of the culture magnesium (Mg2+) concentration below 3.0 mM. The depolymerase is also released after the addition of sublethal concentrations of EDTA to cultures containing 3.0 mM Mg2+. A survey of additional mucoid P. aeruginosa isolates recovered from patients with cystic fibrosis showed that nearly 60% demonstrated similar depolymerase activity while none of the nonmucoid revertants of the parent strains produced detectable depolymerase activity.     

Absence of Escherichia coli O157 in a survey of wildlife from Trinidad and Tobago

Fecal, cloacal, or rectal swabs of free-ranging and captive mammalian and avian wildlife in Trinidad and Tobago were cultured for non-sorbitol fermenting Escherichia coli and tested for O157:H7 strains. Ability of E. coli strains to produce hemolysin and mucoid colonies also was investigated. Of 271 free-ranging mammals tested, 158 (58%) yielded E. coli; only one (< 1%) bacterial isolate was a non-sorbitol fermenter which was not agglutinated by O157 antiserum. All isolates were negative for hemolysin production and mucoid colonial growth. Two hundred and sixty-three (90%) of 293 free-flying birds were positive for E. coli and all isolates were sorbitol fermenters and negative for production of hemolysin and mucoid growth. Of 175 captive wild animals from individual backyard farms and a government demonstration farm, 145 (83%) yielded E. coli with four (2%) non-sorbitol fermenters; all were negative for O157 strains, hemolysin production, and mucoid colonial growth. Of 373 animals in a zoo, 250 (67%) were positive for E. coli with only two (0.5%) non-sorbitol fermenters. All strains were non-hemolytic and non-mucoid farms. It appears that free-ranging and captive avian and mammalian wildlife are not important reservoirs of O157:H7 strains of E. coli in Trinidad and Tobago.     

Alginate biosynthesis in mucoid recombinants of Pseudomonas aeruginosa overproducing GDP-mannose dehydrogenase.

The Pseudomonas aeruginosa algD gene, encoding GDP-mannose dehydrogenase (GMD) and cloned at Chakrabarty's Laboratory in the expression vector pMMB24 (plasmid pVD211), was mobilized into P.aeruginosa strains 8821 and 8821M. Strain 8821M was a high-alginate-producing variant, spontaneously obtained from mucoid strain 8821, with derepressed levels of GMD, a key enzyme in the regulation of alginate biosynthesis, leading to the irreversible oxidation of GDP-mannose to GDP-mannuronic acid. A slight increase in the level of GMD, in both strains harboring the plasmid pVD211 and batch-grown at 37 degrees C without IPTG induction, led to the increase of production rate and the final concentration of alginate produced by control strains harboring the cloning vector. However, the viscosity of the aqueous solutions prepared with the alginate (3 g l-1) produced by mucoid strains harboring pVD211 was lower than those with the alginate produced by the controls (shear rates in the range 0.6-12 s-1). The specific activity of GMD assayed in crude extracts from cells harboring pVD211 and subjected to IPTG induction (0.5 and 3 mM) presented the highest values. However, either the rate of biosynthesis and final concentration of alginate or the viscosity of solutions prepared with the alginate produced by recombinants grown with IPTG were lower than that possible without overproduction. Therefore, the stimulation of the alginate pathway only by manipulating the rate of the step catalysed by GMD, although possible within certain levels, was at the expense of the final exopolysaccharide quality.     

Characterization of mucoid Pseudomonas aeruginosa strains isolated from technical water systems

The occurrence of mucoid Pseudomonas aeruginosa strains was investigated in water samples and surface material from non-clinical aquatic environments. Ten of 81 environmental isolates displayed a mucoid colony type after incubation at 36°C for 24 h on Pseudomonas Isolation Agar. The mucoid strains obtained exclusively from surfaces of technical water systems were characterized in terms of medium-dependent expression of mucoid colonial phenotype, exoenzyme profile, pigment production and O-antigen type. The mucoid strains secreted substantially higher quantities of carbohydrate and uronic acid-containing material compared to non-mucoid environmental isolates. Major slime components of the mucoid strains were identified as O-acetylated alginates that contained higher proportions of mannuronate than guluronate monomer residues and were composed of blocks of poly-mannuronate and poly-mannuronate/guluronate, whereas blocks of poly-guluronate were absent. The results suggest that surfaces in aquatic environments may represent a natural habitat for mucoid (i.e. alginate-overproducing) strains of Ps. aeruginosa with properties similar to clinical mucoid strains.     

Isolation and some properties of extracellular glucan-producing strains of human oral Streptococcus salivarius

A total of eighteen strains of Streptococcus salivarius, which formed rough gelatinous, rough mucoid or smooth mucoid colonies on sucrose agar media, were isolated from the saliva and tongue dorsum of adults. All of the isolates produced glucans as well as fructans from sucrose. The bulk of the glucans was synthesized by the extracellular enzyme fraction and was water insoluble, whereas most of the fructans were synthesized by the cell-associated enzyme fraction and were water soluble. All strains formed microbial deposits on wire and glass surfaces when cultured in sucrose broth, but their sucrose-dependent adhesion was apparently looser than that produced by a cariogenic S. sobrinus strain. The rough gelatinous colony forming strains possessed a greater ability to synthesize water-insoluble glucans and produced heavier deposits with higher cohesion. Preliminary studies showed that the S. salivarius of such characteristic forms of colony were detected primarily in the saliva and tongue dorsum: the smooth mucoid colony formers appeared to predominate in the tongue coat and the rough mucoid and rough gelatinous colony formers were prominent in saliva. Isolation of these S. salivarius from dental plaques was low.     

Survival and Nodulating Ability of Indigenous and Inoculated Rhizobium leguminosarum biovar trifolii in Sterilized and Unsterilized Soil Treated with Sewage Sludge

Rhizobium leguminosarum biovar trifolii was detected in soil from 41 of 47 plots, within nine sewage sludge-treated sites with different soil characteristics and heavy metal contents. However, although population size varied widely, there was no consistent correlation with soil heavy metal concentration. Indigenous populations in 20 plots within four selected sites retained their ability to induce effective nodule formation after incubation of soil in the dark for 165 days. In sterilized (γ-irradiated) soil, Rhizobium survival varied from 0.01% to 95% depending on the soil sample and strain used. Metal-resistant strains with non-mucoid colonies survived less well than mucoid metal-sensitive strains. Received: 26 June 2000/Accepted: 31 July 2000     

Enzymes involved in the biosynthesis of alginate byPseudomonas aeruginosa

Summary Analysis of the enzymes involved in the biosynthesis of alginic acid by mucoidPseudomonas aeruginosa PAO strain's determined the presence of enzymes required to synthesise GDP-mannuronic acid. Addition of polymannuronic acid to an ammonium sulphate precipitate of a cell free alginate suspension indicated the presence of an enzyme which catalysed the epimerisation of mannuronic acid to guluronic acidafter the polymer had been synthesised. The epimerase was shown to be calcium dependant.Various non-mucoid mutants were also studied. The non-mucoid parental strain PAO 381 also contained the enzymes required for alginate synthesis but they were not expressed. Synthesis of alginic acid led to an increase in the level of these enzymes. In the non-mucoid mutants derived from mucoid parents GDP-mannose dehydrogenase was absent in all strains studied. In some of these strains GDP-mannose pyrophosphorylase was also absent, while in other strains, phosphomannase isomerase was absent or greatly reduced.