Exopolysaccharide Slime Research Articles

Early succession of bacterial biofilms in paper machines

Formation of biofilms causes severe problems in paper machines, and hence financial costs. It would be preferable to prevent attachment of the primary-colonizing bacteria than to control the growth of secondary communities, which are sheltered by exopolysaccharide slime layers. We have therefore investigated the early succession of paper-machine biofilms by incubating stainless-steel test coupons in the process water-flow lines in two paper machines operating in slightly alkaline conditions in temperatures (45 and 49 degrees C) supporting thermophilic microbes. Microbial succession was profiled using length heterogeneity analysis of PCR-amplified 16S rRNA genes (LH-PCR) and linking the sequence data of the created 16S rRNA gene libraries to the dominant LH-PCR peaks. Although the bacterial fingerprints obtained from the attached surface communities varied slightly in different samples, the biomarker signals of the dominating primary-colonizing bacterial groups remained high over time in each paper machine. Most of the 16S rRNA gene copies in the early biofilms were assigned to the genera Rhodobacter, Tepidimonas, and Cloacibacterium. The dominance of these sequence types decreased in the developing biofilms. Finally, as phylogenetically identical primary-colonizers were detected in the two different paper mills, the machines evidently had similar environmental conditions for bacterial growth and potentially a common source of contamination.

Pathogenicity of a Highly Exopolysaccharide-producing Halomonas Strain Causing Epizootics in Larval Cultures of the Chilean Scallop Argopecten purpuratus (Lamarck, 1819)

Mass mortalities of larval cultures of Chilean scallop Argopecten purpuratus have repeatedly occurred in northern Chile, characterized by larval agglutination and accumulation in the bottom of rearing tanks. The exopolysaccharide slime (EPS) producing CAM2 strain was isolated as the primary organism from moribund larvae in a pathogenic outbreak occurring in a commercial hatchery producing larvae of the Chilean scallop Argopecten purpuratus located in Bahía Inglesa, Chile. The CAM2 strain was characterized biochemically and was identified by polymerase chain reaction amplification of 16S rRNA as Halomonas sp. (Accession number DQ885389.1). Healthy 7-day-old scallop larvae cultures were experimentally infected for a 48-h period with an overnight culture of the CAM2 strain at a final concentration of ca. 10(5) cells per milliliter, and the mortality and vital condition of larvae were determined by optical and scanning electron microscopy (SEM) to describe the chronology of the disease. Pathogenic action of the CAM2 strain was clearly evidenced by SEM analysis, showing a high ability to adhere and detach larvae velum cells by using its "slimy" EPS, producing agglutination, loss of motility, and a posterior sinking of scallop larvae. After 48 h, a dense bacterial slime on the shell surface was observed, producing high percentages of larval agglutination (63.28 +/- 7.87%) and mortality (45.03 +/- 4.32%) that were significantly (P < 0.05) higher than those of the unchallenged control cultures, which exhibited only 3.20 +/- 1.40% dead larvae and no larval agglutination. Furthermore, the CAM2 strain exhibited a high ability to adhere to fiberglass pieces of tanks used for scallop larvae rearing (1.64 x 10(5) cells adhered per square millimeters at 24 h postinoculation), making it very difficult to eradicate it from the culture systems. This is the first report of a pathogenic activity on scallop larvae of Halomonas species, and it prompts the necessity of an appraisal on biofilm-producing bacteria in Chilean scallop hatcheries.

Vascular Catheter-Related Infection and Sepsis

Background: Catheter-related sepsis is a clinical syndrome characterized by the presence of a catheter-associated infection along with a systemic inflammatory response. The continual increase in the use of central venous catheters (CVCs) has been associated with a substantial risk of infectious complications that prolong the hospital stay and increase costs. Methods: The literature on CVCs was reviewed to determine the incidence of catheterrelated sepsis, its diagnosis, and the role of biofilms in pathogenesis. Results: The European Sepsis Group recently reported that 28% of CVC infections in intensive care unit patients were associated with sepsis, 24% with severe sepsis, and 30% with septic shock. Clinical diagnosis remains difficult. After CVC insertion, the intravascular portion of the device is covered rapidly by a thrombin layer, rich in host-derived proteins, that forms a conditioning film and promotes surface adherence of microbial colonizers. These microorganisms then enter their sessile mode of growth, secreting an exopolysaccharide slime within which organism density is regulated by quorum-sensing molecules. Microorganisms are dispersed in clumps that can become septic emboli. Antiadhesive, antiseptic, and antibiotic coatings of catheters have demonstrated only modest clinical efficacy. Conclusion: Our group is involved in the design and in vitro assessment of new polymeric matrices for controlled release of antimicrobial molecules, and in comparative clinical studies of conventional versus antibiotic-coated or -impregnated catheters.

Vascular Catheter-Related Infection and Sepsis

Catheter-related sepsis is a clinical syndrome characterized by the presence of a catheter-associated infection along with a systemic inflammatory response. The continual increase in the use of central venous catheters (CVCs) has been associated with a substantial risk of infectious complications that prolong the hospital stay and increase costs. The literature on CVCs was reviewed to determine the incidence of catheterrelated sepsis, its diagnosis, and the role of biofilms in pathogenesis. The European Sepsis Group recently reported that 28% of CVC infections in intensive care unit patients were associated with sepsis, 24% with severe sepsis, and 30% with septic shock. Clinical diagnosis remains difficult. After CVC insertion, the intravascular portion of the device is covered rapidly by a thrombin layer, rich in host-derived proteins, that forms a conditioning film and promotes surface adherence of microbial colonizers. These microorganisms then enter their sessile mode of growth, secreting an exopolysaccharide slime within which organism density is regulated by quorum-sensing molecules. Microorganisms are dispersed in clumps that can become septic emboli. Antiadhesive, antiseptic, and antibiotic coatings of catheters have demonstrated only modest clinical efficacy. Our group is involved in the design and in vitro assessment of new polymeric matrices for controlled release of antimicrobial molecules, and in comparative clinical studies of conventional versus antibiotic-coated or -impregnated catheters.

Dilute and semi-dilute solution properties of an exopolysaccharide from Escherichia coli strain S61

The solution properties of colanic acid, an exopolysaccharide slime isolated from Escherichia coli strain 61, have been investigated. Previous chemical analysis ( Sutherland, 1969) had shown that this polysaccharide contained glucose, galactose and glucuronic acid, in addition to acetate and pyruvate. Three batches (S61-1, S61-2 and S61-3) of colanic acid were used for rheological measurements. The intrinsic viscosity of S61-1, S61-2 and S61-3 were found to be 20.8±0.4 dl/g (0.2 M NaCl), 22.7±0.6 dl/g (0.2 M NaCl) and 21.1±0.3 dl/g (0.2 M CaCl 2) respectively, which indicates that the colanic acid is of high molecular weight. The semi-dilute solution characteristics, investigated by steady shear and dynamic rheometry, suggest that colanic acid acts as a well behaved polymer entanglement system. The viscosity-polymer concentration relationship of colanic acid shows no significant ‘critical’ breakpoint concentration on passing from a dilute to a semi-dilute solution. Instead, there seems to be a gradual change in slope from the first regime, ∼1, to the second regime, ∼3.6.

In vitro adherence and accumulation of Staphylococcus epidermidis RP 62 A and Staphylococcus epidermidis M7 on four different bone cements.

Bacterial resistance of Staphylococcus epidermidis, a serious pathogen of implant-related infections, to antibiotics is related to the production of a glycocalyx slime that impairs antibiotic access and the killing by host defense mechanisms. In vitro studies of different bone cements containing antibiotics, developed for the prevention of biomaterial-associated infection, could not always demonstrate complete eradication of biomaterial-adherent bacteria. We have investigated four different bone cements in regard to bacterial accumulation of a slime-producing strain RP 62 A and its isogenic mutant M7 lacking the ability to produce exopolysaccharide slime using a bacterial adhesion assay and modified Kirby-Bauer technique. A significant effect of exopolysaccharide production for the accumulation on bone cement could be demonstrated. The gentamicin/clindamycin bone cement was the only tested biomaterial that produced a large zone of bacterial inhibition in the inoculated area adjacent to the biomaterial. The bacterial adhesion was not reduced significantly and there was no correlation between zones of inhibition on blood agar plates and the quantitative adhesion assay. The clinical efficacy of the gentamicin/clindamycin bone cement must be proven in vivo.

SECRETION OF EXTRACELLULAR VIRULENCE FACTORS BY PLANT PATHOGENIC BACTERIA

Many bacterial plant pathogens produce products that contribute to virulence (21). What constitutes an virulence factor? In prin­ ciple, any molecule presented on the bacterial cell surface or translocated to the environment could act as a virulence determinant if it influ­ ences growth of the pathogen in planta. Loosely defined in this fashion, extracellular virulence factors include plant cell wall-degrading enzymes, toxins, DNA, hormones, siderophores, and signaling molecules (e.g. see 9, 15, 21, 27, 49, 50). Also, cell surface-anchored structures such as pili, flagella, lipopolysaccharide, exopolysaccharide slime layers, and outer membrane pro­ teins could have roles in bacterial survival within, or ingression of, the host plant (e.g. see 9, 18, 24). With such a broad definition of extracellular virulence factors it is not possible to cover the corresponding transport pro­ cesses involved in each case in this review. Consequently, I concentrate on a review of the secretion of a selection of molecules, particularly proteins, which are targeted totally outside of the bacterial cell, and will generally exclude discussion of the cell surface-anchored/associated structures, except in isolated cases. In addition, I concentrate exclusively on gram-negative bacterial plant

Antibiotic impregnated orthopaedic bone cement: Kinetics and impact of teicoplanin on staphylococcal colonization

Previous studies in our laboratory have questioned the efficacy of antibiotic impregnated bone cement to prevent staphylococcal colonization of orthopaedic implants following surgery. Teicoplanin a glycopeptide antibiotic with excellent anti-staphylococcal activity was added to polymethylmethacrylate in concentrations ranging from 2-5 wt%. Five staphylococcal strains recovered from orthopaedic implant infections were chosen for study: 2-Staphylococcus aureus and 3-Staphylococcus eoidermidis. Two of the staphylococcal isolates were identified as producing an exopolysaccharide slime following alcian blue staining with lysine. The in vitro release of antibiotic from the resin surface was determined by biological assay at 0, 12, 24, 48, 96 and 10 days post-study. Mean teicoplanin concentrations ranged from 200 to 430 ug/ml at 0 hour and 22 to 50 ug/ml at 10 days post-study. Teicoplanin impregnated and drug free resin controls were exposed to standardized inoculum (5.0 log10 colony forming units/ml) of the staphylococcal clinical isolates and incubated at 35°C in physiologic buffered saline with 0.25% dextrose.

Decomposition of exopolysaccharide slime by a bacteriophage enzyme

A bacterial virus-encoded endodepolymerase has been shown to decompose exopolysaccharide slime produced by its sewage sludge-derived bacterial host. Substantial viscosity reduction and increased centrifugal compaction of a single slime-producing microbial slurry were achieved on enzyme addition. This demonstrates the feasibility of improving mechanical dewatering of biological sludges by use of enzymes in which the slime entrapped water is released and becomes more readily available for separation from the sludge solids matrix.

Gram-positive bacteria: an overview and summary of session.

The more pathogenic gram-positive bacteria present a complex array of surface structures to the human or animal host. The cell wall of Staphylococcus aureus has a pattern of surface proteins; the predominant one is protein A. Virulent S. aureus strains may also produce polysaccharide capsules in vivo that impede opsonization and phagocytosis in the absence of anticapsular antibody. Coagulase-negative staphylococci commonly elaborate an exopolysaccharide slime that may promote adherence to plastic surfaces and interfere with host responses. Structure-function relationships for some antiphagocytic M proteins of group A streptococci are now well understood, and recombinant techniques offer the prospect of multivalent vaccines. The best known surface protein of group B streptococci is the c (Ibc) protein, which stimulates protective antibody in animals and may be an important virulence factor. Monoclonal antibodies to types Ib, II, and III group B streptococci have also confirmed the presence of multiple immunodeterminants on these antiphagocytic polysaccharides. A protein on the surface of pneumococci has been shown to induce protective antibody and to enhance pneumococcal virulence in mice, suggesting a potential alternative or adjunct to pneumococcal polysaccharide vaccines. Listeria also possess a variety of cell surface structures important in pathogenesis. Surface components are, therefore, critical determinants of the interaction of gram-positive bacteria with the host.

The role of germination in sorghum reconstitution

Commercial reconstitution of sorghum for cattle feed was simulated in the laboratory, with partial germination of the grain prior to anaerobic storage as an added variable. After steeping to 30–35% moisture, samples of red hybrid sorghum grain were allowed to germinate for up to 2 days prior to anaerobic storage periods of 0–21 days. Changes in nitrogen and carbohydrate solubility, reducing sugars, enzyme activity, dry matter loss and particle size index were determined. Chemical and physical modification of the grain appeared to be minimal during anaerobic storage when it was not preceded by germination. Significantly higher nitrogen and carbohydrate solubility were achieved in 6 days (1 day germination + 5 days storage) than in 21 days of strictly anaerobic storage. The rapid incorporation of soluble nitrogen and carbohydrate into bacterial cell mass and exo-polysaccharide slime appeared to limit the increases in dry-matter loss resulting from germination. Protease activity resulted from both germination and bacterial growth, while amylase activity could be attributed only to germinating seeds. Reductions in particle size index could be attributed to steeping, germination and storage effects, the largest component appearing to result from steeping. It was concluded that the onset of germination can greatly accelerate the beneficial bacterial fermentation occurring during sorghum reconstitution, probably by increasing bacterial growth rates and surface area accessible to bacterial action.

Influence of anti-slime glycolipoprotein serum on the interaction between Pseudomonas aeruginosa and macrophages.

Glycolipoprotein, a purified fraction of the exopolysaccharide slime of Pseudomonas aeruginosa, was identified as responsible for a number of the biological activities of viable cells, including toxicity and immunogenicity capable of stimulating protective antibody against the lethal effects of viable cells. Antiserum against glycolipoprotein also mediated the phagocytosis and subsequent killing of viable P. aeruginosa by unstimulated mouse peritoneal macrophages. In the absence of anti-glycolipoprotein serum, macrophages did not significantly reduce the number of bacteria. The presence of complement in the experimental mixture did not affect the reduction of bacteria by the macrophage in the presence of anti-glycolipoprotein serum. The limiting effect of antiserum concentration on macrophage activity was studied, and maximal activity was found at 2%, with no further increase in activity at 5% Preopsonization of the bacteria with anti-glycolipoprotein serum had little effect on the course of phagocytosis within the experimental conditions. Variations in bacterium-to-macrophage input ratios, ranging from 30:1 to 1:30, did not affect the capacity of the macrophages for phagocytosis.

Exopolysaccharide colanic acid and its occurrence in the Enterobacteriaceae.

A study of strains from the genera Salmonella, Escherichia, and Aerobacter has shown that under appropriate conditions many strains produce an exopolysaccharide slime of identical composition, which has been identified as colanic acid on the basis of its chemical composition and its sensitivity to certain bacteriophage-induced depolymerase enzymes. Chemical analysis shows that the polysaccharide contains O-acetyl groups in addition to the sugars glucose, galactose, fucose, and glucuronic acid. Mild acid hydrolysis has led to the isolation of a beta-glucosylfucose in addition to glucuronic acid containing oligosaccharides. Many strains were found to synthesize colanic acid under normal conditions of growth or under conditions favoring polysaccharide synthesis, whereas others only synthesized colanic acid when the control mechanism was derepressed by p-fluorophenylalanine.

Structural studies on colanic acid, the common exopolysaccharide found in the enterobacteriaceae, by partial acid hydrolysis. Oligosaccharides from colanic acid.

The exopolysaccharide slime colanic acid has been isolated from representative strains of Escherichia coli, Salmonella typhimurium and Aerobacter cloacae. Analysis showed that each polymer contained glucose, galactose, fucose and glucuronic acid, together with acetate and pyruvate. The molar proportions of these components were 1:1.8:1.9:1:1:1 approximately. On the basis of periodate oxidation of the natural and deacetylated polysaccharide, glucose is proposed as the site of the acetyl groups. The pyruvate is attached to galactose. Three neutral oligosaccharides and ten electrophoretically mobile oligosaccharides were isolated and partially characterized. Four of the fragments were esters of pyruvic acid. Most oligosaccharides were isolated from all three polysaccharide preparations. Three further oligosaccharides were isolated from carboxyl-reduced colanic acid and sodium borotritide was used to label the glucose derived from glucuronic acid in these fragments. One trisaccharide was obtained from periodate-oxidized polysaccharide. On the basis of these oligosaccharides a repeating hexasaccharide unit of the following structure is proposed: [Formula: see text] The significance of this structure in colanic acid biosynthesis is discussed.

The exopolysaccharide of Klebsiella aerogens A3 (S1) (type 54). The isolation of O-acetylated octasaccharide, tetrasaccharide and trisaccharide.

The exopolysaccharide slime produced by Klebsiella aerogenes A 3 (Sl) (type 54) is an O-acetylated polysaccharide, the components of which are glucose, glucuronic acid, fucose and acetyl in the molar proportions 4:2:2:1. A phage-induced fucosidase was obtained that hydrolyses the polysaccharide to give an octasaccharide having the same constituents in the same molar proportions. This octasaccharide (O3) is considered to be the repeating unit of the polysaccharide. It is hydrolysed by other phage-induced fucosidases described earlier to release two tetrasaccharides (O1 and O2). These differ only in that tetrasaccharide O2 is acetylated. An acetylated trisaccharide of structure beta-glucosylglucuronosylfucose was prepared from tetrasaccharide O2. A further unidentified group is present. Cell-free preparations were used to acetylate the disaccharide alpha-glucuronosylfucose. From these results the structure of the octasaccharide (O3) is postulated and its significance in the biosynthesis of the polysaccharide discussed.