Partial spectrum of microorganisms found in dentures and possible disease implications.

While it would appear that denture surfaces alone become colonized by microorganisms, this study showed that the porosity of denture material allows for contamination throughout the entire denture. Further, the numerous opportunistic and pathogenic microorganisms found in this study were unexpected and are known to produce not only substantial oral infections, but also systemic diseases.

Comparison of the effectiveness of several denture sanitizing systems: a clinical study.

The purpose of this clinical study was to test the effectiveness of three methods of decontamination on complete dentures. Dentures worn by patients for varying lengths of time were handled aseptically and treated with three different treatment modalities. The dentures were touched and sectioned and then retouched to a variety of microbiological media. The quantity of microbial growth was recorded and predominating microorganisms were identified using standard microbiological techniques. System A was found to consistently decontaminate and sanitize dentures worn by patients. System B and System C showed variable reduction of microorganisms. An unexpected spectrum of both pathogenic and opportunistic microorganisms was found in the dentures examined, including a wide range of gram-negative bacteria, gram-positive bacteria, and yeasts. A wide range of microorganisms must be considered when treating either oral or systemic diseases in denture wearers. Denture hygiene and decontamination are critical to the prevention of oral and systemic disease transmission. The dentures of ill patients must be considered as possible sources of pathogenic microorganisms.

The occurrence of 4-amino-4-deoxy-arabinose in LPS of supersusceptible strain Pseudomonas aeruginosa Z61: noncorrelation with polymyxin resistance.

Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities. The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P. aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC). Z61 LPS (compared with wild-type strain PAO1) has reduced amounts of rhamnose and higher concentrations of hydroxy fatty acids, 4-AraN, and phosphates. 31P Nuclear magnetic resonance revealed that Z61 LPS phosphates are configured in monophosphates, phosphodiesters, pyrophosphomonoesters, and glycosidic pyrophosphodiester groups. The presence of 4-AraN in P. aeruginosa LPS did not correlate with antimicrobial resistance.

Biochemical characterization of lipopolysaccharides extracted from a hydrophobic strain of Pasteurella multocida.

Lipopolysaccharides were extracted from freeze-dried cells of Pasteurella multocida strain P-1581 (serotype 8) by the phenol-chloroform-petroleum ether method and biochemically analysed using standard procedures. The primary neutral sugars were glucose, galactose and heptose. No deoxy sugars were detected. Amino sugars included galactosamine, glucosamine and glucosamine-6-phosphate. 3-Deoxy-D-manno-2-octulosonic acid was present at a relatively low concentration. The analyses included identification and quantification of phosphate and alanine. The primary fatty acids and their approximate relative ratios were 3-hydroxytetradecanoate and tetradecanoate 2:1. Tetradecanoic acid was bound almost exclusively by ester linkages. 3-Hydroxytetradecanoic acid was bound primarily by amide linkages, although significant numbers of ester-bound residues were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses indicated that the lipopolysaccharides were of low molecular weight.

Antibodies from chronically infected cystic fibrosis patients react with lipopolysaccharides extracted by new micromethods from all serotypes of Pseudomonas aeruginosa.

Micromethods were developed to extract lipopolysaccharide (LPS, endotoxin) from single bacterial colonies of the 20 recognized Pseudomonas aeruginosa type strains. The appearance of these LPSs in polyacrylamide gel electrophoresis (PAGE) and their reactivity with serum of cystic fibrosis (CF) patients chronically infected with P. aeruginosa was studied. Silver staining of LPS after PAGE showed that 13 of the P. aeruginosa LPSs had high numbers of O-repeating units arranged in 1-4 clusters of banding. Low-molecular-weight LPS fractions were more prominent in six of the serotype strains, of which O:7 and O:14 appeared semi-rough. Corresponding immunoblots using the CF sera showed LPS patterns very similar to the silver-stained appearance, indicating an immune reaction to all P. aeruginosa LPS including that from the newly discovered O:18, O:19 and O:20. This was unexpected since only a few serotype strains (mostly O:3, O:6 and O:9) had been isolated from the patients. Absorption experiments using purified and chemically defined P. aeruginosa rough LPS and smooth LPS suggested these immune reactions were due to antibodies cross-reactive to core/lipid A as well as to lower molecular weight O-polysaccharides or "A-bands". However, in some cases O:3, O:6, and O:9 LPSs were also found to contain additional distinct O-epitopes. Immune recognition of various polyagglutinable P. aeruginosa LPSs seemed also to be caused by cross-reactive antibodies. The described microextraction methods, followed by PAGE and silver staining or immunoblotting, are easy and convenient techniques with which to study antibodies against LPS epitopes and to screen for LPS phenotypic appearance using only a few bacterial colonies from larger numbers of Gram-negative bacterial strains.

Relationship between chemical composition and biological function of Pseudomonas aeruginosa lipopolysaccharide: effect on human neutrophil chemotaxis and oxidative burst.

There are conflicting data on the effect of bacterial lipopolysaccharides (LPS) on the function of human neutrophils. The present study was designed to examine the relationship between chemical composition and the modulatory effect of LPS on human neutrophil function. LPS was extracted from five strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients by the hot phenol-water method. Chemical characterization included neutral sugars, amino components, and fatty acids. Neutrophils isolated from peripheral blood of healthy individuals were preincubated with different concentrations of LPS. After preincubation, the chemotaxis and chemiluminescence of neutrophils to various stimuli were determined. It was shown that LPS from different strains did not exert the same degree of regulatory effect on neutrophil functions. LPS from strain 174-O:9 exerted the most pronounced effect on neutrophil function seen as inhibition of neutrophil chemotaxis toward the chemotactic peptide f-Met-Leu-Phe and zymosan-activated serum (ZAS) and priming of the cells for less than or equal to 8-fold enhancement of chemiluminescence response to f-Met-Leu-Phe. Conversely, LPS from strain 1118-O:3 had no effect on neutrophil chemotaxis and a slight effect on chemiluminescence. The major differences in chemical composition of the LPS from these two strains are in the rhamnose and heptose content of the O side chain and in the alanine content of the core region. These data indicate that chemical composition of the LPS molecule may play an important role in biological activity of LPS.

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.

Fatty acid alterations and polymyxin B binding by lipopolysaccharides from Pseudomonas aeruginosa adapted to polymyxin B resistance.

Lipopolysaccharides were extracted from freeze-dried cells of Pseudomonas aeruginosa PAO1 (polymyxin B susceptible), isolate A (polymyxin B resistant), and isolate A-reverted (polymyxin B intermediate resistance) by either the phenol-chloroform-petroleum ether or the modified phenol-water method. Isolate A and isolate A-reverted had drastic losses of 2-hydroxydodecanoic acid and significant decreases in 3-hydroxydecanoic acid. Concentrations of amide-linked 3-hydroxydecanoic acid were similar in all three strains. Minor alterations were noted in the composition of 3-deoxy-D-manno-2-octulosonic acid, heptose, phosphate, neutral sugars, and amino compounds. The concentrations of rhamnose in isolate A and of rhamnose and glucose in isolate A-reverted were significantly different from those in PAO1. Trace amounts of mannose and other minor unidentified carbohydrates were detected in all strains. Polymyxin B included in isolate. A growth medium complexed with lipopolysaccharides and remained bound throughout purification. PAO1 lipopolysaccharides bound more polymyxin B than did isolate A lipopolysaccharides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated minor differences in smooth- and rough-form lipopolysaccharides of the different strains. We propose that loss of hydroxy fatty acids from lipopolysaccharides perturbs outer membrane hydrophobicity and is a contributing factor to polymyxin B adaptive resistance.

Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Antibodies to Pseudomonas aeruginosa from 10 cystic fibrosis patients with chronic P. aeruginosa lung infections were quantitatively and qualitatively analyzed. The development of specific antibodies in patient serum was evaluated in a longitudinal study (1972 to 1987). The concentrations and specificities of immunoglobulin G (IgG) and IgM antibodies to purified lipopolysaccharides (LPS) from clinical isolates of P. aeruginosa and to a variety of other gram-negative bacteria were studied by immunoblotting and enzyme-linked immunosorbent assay techniques. Results were compared with the number of immunoprecipitates to P. aeruginosa whole-cell extracts detected by crossed immunoelectrophoresis. IgG, but not IgM, anti-Pseudomonas LPS concentrations increased significantly at the onset of chronic infection and continued to increase during the course of the infection. There was a good positive correlation between the concentration of IgG anti-Pseudomonas LPS antibodies and the number of crossed-immunoelectrophoresis precipitins. The increases in IgG anti-LPS antibody concentrations were much higher to Pseudomonas LPS than to other LPSs. Binding studies demonstrated an increase in binding of IgG anti-Pseudomonas LPS during infection, whereas the binding of other anti-LPS antibodies decreased. Immunoblotting studies confirmed that antibodies reacted strongly with Pseudomonas LPS and weakly with Escherichia coli core-lipid A. The specificity of the reaction with Pseudomonas LPS increased with the duration of infection. It is concluded that anti-LPS response in cystic fibrosis patients during chronic P. aeruginosa infection demonstrates a marked increase in IgG anti-Pseudomonas LPS antibody concentration, specificity, and affinity. The anti-LPS enzyme-linked immunosorbent assay is proposed as a routine test to diagnose and to follow the course of chronic P. aeruginosa lung infection in patients with cystic fibrosis.

Comparative immunochemistry of lipopolysaccharides from typable and polyagglutinable Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis.

Lipopolysaccharide (LPS) was extracted and purified from three Pseudomonas aeruginosa strains isolated from the infected lungs of patients with cystic fibrosis. Two of the strains could be typed by O-specific antibody (O:3 and O:9), and the third was polyagglutinable (O:3/9). The separated LPS was characterized by chemical and serological methods. The main neutral sugar constituents (glucose, rhamnose, and heptose) were found in various proportions in the three strains, whereas the amounts of glucosamine, galactosamine, ketodeoxyoctonate, and phosphate were more constant. Ester-bound C12, C16, 3-OH-C10, and 2-OH-C12, together with amide-bound 3-OH-C12, fatty acids were present in equimolar proportions in all three strains. Considerable amounts of LPS were liberated in the culture supernatant of the O:3 bacteria but not in those from the other two strains. This free LPS was shown to be immunologically identical to the cell-bound LPS and the extracted LPS. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, O:3 and O:9 LPS showed a ladder pattern characteristic of smooth LPS, while O:3/9 LPS appeared rough. Rabbit antisera used for O-typing were found by enzyme-linked immunosorbent assay to contain anti-LPS antibodies that reacted strongly with homologous LPS, moderately with O:3/9 LPS, and slightly with heterologous LPS. Immunoblotting showed that common antigenic determinants in the core-lipid A part were involved in the observed cross-reaction. The polyagglutinability of P. aeruginosa may be explained by the antibodies to these common determinants that arose from the partial absence of O polysaccharides.

Chemical alterations in cell envelopes of Pseudomonas aeruginosa upon exposure to polymyxin: a possible mechanism to explain adaptive resistance to polymyxin.

Polymyxin-susceptible cells of Pseudomonas aeruginosa were exposed for 10, 30, and 60 min to growth medium containing 6000 units of polymyxin per millilitre. Exposure for 10 min resulted in lipid alterations in the cell envelope. A large reduction in the content of both phosphatidylethanolamine and phosphatidylglycerol with a large increase in both diphosphatidylglycerol and free fatty acids was found upon analysis by thin-layer chromatography. The cellular percentage of readily extractable lipid (REL) was reduced, and the phospholipid proportion of the REL decreased with polymyxin exposure. Polymyxin exposure for 30 and 60 min only slightly enhanced these chemical alterations. The cell envelope alterations found were characteristic for strains which become adaptively resistant to polymyxin. Treatment of the cells with chloramphenicol or KCN prior to polymyxin exposure did not prevent the lipid alterations from occurring. These observations suggest that the polymyxin-susceptible cell population adapts to polymyxin resistance by the rapid alteration of the cell envelopes of the entire cell population. We propose the theory that cell envelope phospholipases and proteases play a major role in the adaptive response and that the cation content of the cell envelope may be a critical controlling factor in the process.

Conversion of phospholipids to free fatty acids in response to acquisition of polymyxin resistance in Pseudomonas aeruginosa.

The readily extractable lipids from a Pseudomonas aeruginosa isolate stepwise adapted to polymyxin resistance were compared with those of the susceptible parent and of a revertant strain which regained susceptibility. Significant qualitative and quantitative lipid alterations accompany the acquisition of resistance. Changes include the appearance of a major unidentified lipid (lipid X) unique to the readily extractable lipids of resistant cells. Comparative studies with parent and revertant strains indicated a significant decrease in the phospholipid content of resistant cells. Thin-layer chromatography of resistant-cell readily extractable lipids demonstrated: (i) the emergence of lipid X (36% of total readily extractable lipids), (ii) a decrease in phosphatidylethanolamine and phosphatidylglycerol, and (iii) an increase in diphosphatidylglycerol. Lipid X was purified by preparative silicic acid column chromatography and thin-layer chromatography and characterized by analytical thin-layer chromatography, column adsorption chromatography, and gas-liquid chromatography. Data from this study indicated that lipid X was a mixture of free fatty acids. The fatty acids present in lipid X were qualitatively and quantitatively the same as the fatty acids esterified to the phospholipids in the readily extractable lipids.

Chemical alterations in cell envelopes of polymyxin-resistant mutants of Pseudomonas aeruginosa grown in the absence or presence of polymyxin.

The polymyxin-resistant mutant strains H181 and H185 of Pseudomonas aeruginosa, after growth in the absence or presence of polymyxin, were compared with the polymyxin-sensitive H103 strain as to their cell envelope protein composition (determined by slab polyacrylamide gel electrophoresis) and their lipid composition. When grown in the absence of polymyxin, the H181 and H185 strains had an increased content of the outer membrane protein H1 with a decreased content of the outer membrane proteins D2 and F. After growth in the presence of polymyxin, the content of H1, D2, and F were all decreased. Significant alterations in the lipid composition of the H181 and H185 strains were found after growth in the absence of polymyxin. These lipid alterations were enhanced upon growth in the presence of polymyxin, with both strains having a reduced content of phosphatidylethanolamine and phosphatidylglycerol and an increased content of diphosphatidylglycerol and an unidentified lipid thought to be a neutral lipid. Other workers have proposed that an increased content of H1 protein is the molecular basis for polymyxin resistance in the H181 and H185 strains. Our observations make this hypothesis appear unlikely.

Lipid alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

The lipid composition of cells of Pseudomonas aeruginosa strains resistant to polymyxin was compared with the lipid composition of cells of polymyxin-sensitive strains as to their content of readily extractable lipids (RELs), acid-extractable lipids, the fatty acid composition of RELs, and the contents of various phospholipids in the REL fraction. The polymyxin-resistant strains had an increased content of RELs, but a decreased phospholipid content. The REL fraction from the polymyxin-resistant strains had an increased content of unsaturated fatty acids accompanied by a decreased content of cyclopropane fatty acids as compared with the fatty acid composition of RELs from polymyxin-sensitive strains. The phosphatidylethanolamine content was greatly reduced in the polymyxin-resistant strains, whereas the content of an unidentified lipid, thought to be a neutral lipid lacking either a phosphate, free amino, or choline moiety, was greatly increased. Cell envelopes of the polymyxin-resistant strains contained reduced concentrations of Mg2+ and Ca2+ as compared with the cell envelopes of polymyxin-sensitive strains. It appears that polymyxin resistance in these strains is associated with a significant alteration in the lipid composition and divalent cation content of the cell envelope.

Effects of carbon sources on chemical composition of cell envelopes of Pseudomonas aeruginosa in association with polymyxin resistance.

Cells of Pseudomonas aeruginosa 015 were grown in basal medium with isobutyrate, DL-2-methylbutyrate, isovalerate, L-valine, L-isoleucine, L-leucine, D-glucose, or L-glutamate as the carbon source. Their resultant susceptibility to polymyxin B varied from a minimal inhibitory concentration of 2 U of polymyxin per ml for isobutyrate-grown cells to 975 U/ml for L-glutamate-grown cells. Cell envelopes from cells grown with each carbon source were compared with cell envelopes from cells grown in Mueller-Hinton broth as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. No pattern of cell envelope content of total protein, carbohydrate, 2-keto-3-deoxyoctonate, or outer membrane protein concentrations could be correlated with the degree of resistance to polymyxin. In these cells increased resistance to polymyxin was not associated with the loss of outer membrane proteins and lipopolysaccharide by the cell envelope.

Effects of carbon sources on antibiotic resistance in Pseudomonas aeruginosa.

The metabolism of branched-chain amino acids, branched-chain acyl derivatives, d-glucose, l-glutamate, and Mueller-Hinton medium was investigated to determine their effects on the growth, lipid composition, and antibiotic susceptibility of Pseudomonas aeruginosa. The unsaturated fatty acid content of the readily extractable lipids was altered by growth on selected branched-chain amino acids and their acyl derivatives. Bacteria grown on branched-chain acyl derivatives became more susceptible to polymyxin B and colistin. The effect acyl derivatives had on increasing susceptibility was also manifest in mixed media which contained both an acyl derivative and a carbon source which did not increase susceptibility. Growth on branched-chain amino acids gave mixed results which were dependent on a number of factors, including unique manifestations of individual amino acids, growth conditions, and availability of other carbon sources. The cultural conditions which altered susceptibility to polymyxin antibiotics did not correlate with similar effects on susceptibility to carbenicillin and gentamicin. An adaptive resistance to polymyxin B was observed when the sole carbon source was d-glucose or l-glutamate.

The role of enoyl-coa hydratase in the metabolism of isoleucine by Pseudomonas putida.

The purpose of the present study was to determine if the enoyl coenzyme A hydratase formed by Pseudomonas putida during growth on isoleucine was a unique enzyme specific for isoleucine metabolism. The highest levels of the hydratase were formed during growth on isoleucine intermediates and the lowest levels during growth on glutamate and glucose. Data from growth experiments revealed that 2-methyl-3-hydroxybutyryl coenzyme A hydratase, an enzyme unique to isoleucine metabolism and enoyl coenzyme A hydratase were coordinately induced, but that 3-hydroxyacyl coenzyme A dehydrogenase was under separate control. The hydratase was purified 180-fold from isoleucine cells, and its physical and catalytic properties reported. The highest activity was with crotonyl coenzyme A,Vmax = 1100 x 10(3) moles/min mole enzyme, next was tiglyl coenzyme A, Vmax = 61 x 10(3) moles/min mole enzyme, and last was 3-methyl-crotonyl coenzyme A, Vmax = 2.3 x 10(3) moles/min mole enzyme. Enzyme purified from butyrate cells had the same elution patterns during column chromatography and catalytic properties as the enzyme from isoleucine cells. These data support the conclusion that a single enzyme in P. putida is responsible for the hydration of both tiglyl coenzyme A and crotonyl coenzyme A.