Impact of cranberry juice on initial adhesion of the EPS producing bacterium Burkholderia cepacia

The impact of cranberry juice was investigated with respect to the initial adhesion of three isogenic strains of the bacterium Burkholderia cepacia with different extracellular polymeric substance (EPS) producing capacities, viz. a wild-type cepacian EPS producer PC184 and its mutant strains PC184rml with reduced EPS production and PC184bceK with a deficiency in EPS production. Adhesion experiments conducted in a parallel-plate flow chamber demonstrated that, in the absence of cranberry juice, strain PC184 had a significantly higher adhesive capacity compared to the mutant strains. In the presence of cranberry juice, the adhesive capacity of the EPS-producing strain PC184 was largely reduced, while cranberry juice had little impact on the adhesion behavior of either mutant strain. Thermodynamic modeling supported the results from adhesion experiments. Surface force apparatus (SFA) and scanning electron microscope (SEM) studies demonstrated a strong association between cranberry juice components and bacterial EPS. It was concluded that cranberry juice components could impact bacterial initial adhesion by adhering to the EPS and impairing the adhesive capacity of the cells, which provides an insight into the development of novel treatment strategies to block the biofilm formation associated with bacterial infection.     

Role of cranberry juice on molecular-scale surface characteristics and adhesion behavior of Escherichia coli

Cranberry juice has long been believed to benefit the prevention and treatment of urinary tract infections (UTIs). As the first step in the development of infection, bacterial adhesion is of great research interest, yet few studies have addressed molecular level adhesion in this context. P-fimbriated Escherichia coli play a major role in the development of a serious type of UTI, acute pyelonephritis. Experiments were conducted to investigate the molecular-scale effects of cranberry juice on two E. coli strains: HB101, which has no fimbriae, and the mutant HB101pDC1 which expresses P-fimbriae. Atomic force microscopy (AFM) was used to investigate both bacterial surface characteristics and adhesion forces between a probe surface (silicon nitride) and the bacteria, providing a direct evaluation of bacterial adhesion and interaction forces. Cranberry juice affected bacterial surface polymer and adhesion behavior after a short exposure period (<3 h). Cranberry juice affected the P-fimbriated bacteria by decreasing the adhesion forces between the bacterium and tip and by altering the conformation of the surface macromolecules on E. coli HB101pDC1. The equilibrium length of polymer (P-fimbriae) on this bacterium decreased from approximately 148 to approximately 48 nm upon being exposed to cranberry juice. Highly acidic conditions were not necessary for the prevention of bacterial adhesion, since neutralization of cranberry juice solutions to pH = 7.0 allowed us to observe differences in adhesion between the E. coli strains. Our results demonstrate molecular-level changes in the surfaces of P-fimbriated E. coli upon exposure to neutralized cranberry juice.     

Impact of an extracellular polymeric substance (EPS) precoating on the initial adhesion of Burkholderia cepacia and Pseudomonas aeruginosa

Extracellular polymeric substances (EPS) significantly influence bacterial adhesion to solid surfaces, but it is difficult to elucidate the role of EPS on bacterial adhesion due to their complexity and variability. In the present study, the effect of EPS on the initial adhesion of B. cepaciaepacia PC184 and P. aeruginosa PAO1 on glass slides with and without an EPS precoating was investigated under three ionic strength conditions. The surface roughness of EPS coated slides was evaluated by atomic force microscopy (AFM), and its effect on initial bacterial adhesion was found to be trivial. X-ray photoelectron spectroscopy (XPS) studies were performed to determine the elemental surface compositions of bacterial cells and substrata. The results showed that an EPS precoating hindered bacterial adhesion on solid surfaces, which was largely attributed to the presence of proteins in the EPS. This observation can be attributed to the increased steric repulsion at high ionic strength conditions. A steric model for polymer brushes that considers the combined influence of steric effects and DLVO interaction forces is shown to adequately describe bacterial adhesion behaviors.     

Starvation-specific formation of a peripheral exopolysaccharide by a marine Pseudomonas sp., strain S9.

The marine bacterium Pseudomonas sp. strain S9 produces exopolysaccharides (EPS) during both growth and total energy source and nutrient starvation. Transmission electron microscopy of immunogold-labeled cells demonstrated that the EPS is closely associated with the cell surface during growth (integral EPS), while both the integral form and a loosely associated extracellular (peripheral) form were observed during starvation. Formation and release of the latter rendered the starvation medium viscous. In addition, after 3 h of starvation in static conditions, less than 5% of the cells were motile, compared with 100% at the onset of starvation and approximately 80% subsequent to release of the peripheral EPS at 27 h of starvation. Inhibition of protein synthesis with chloramphenicol added before 3 h of starvation caused no increase in viscosity. However, addition of chloramphenicol at 3 h did not prevent the subsequent increase in viscosity displayed by S9 cells. The amount of integral EPS increased for both nontreated and chloramphenicol-treated S9 cells during the first hour of starvation, with a subsequent equal decrease. The chloramphenicol-treated cells, as well as cells of a transposon-generated mutant strain deficient in peripheral EPS formation, remained adhesive to a hydrophobic inanimate surface during the initial 5 h of starvation, whereas nontreated wild-type cells had progressively decreased adhesion capacity. During the initial 5 h of starvation, most of the nontreated cells but only a small fraction of the chloramphenicol-treated and mutant cells detached from the hydrophobic substratum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Starvation-specific formation of a peripheral exopolysaccharide by a marine Pseudomonas sp., strain S9

The marine bacterium Pseudomonas sp. strain S9 produces exopolysaccharides (EPS) during both growth and total energy source and nutrient starvation. Transmission electron microscopy of immunogold-labeled cells demonstrated that the EPS is closely associated with the cell surface during growth (integral EPS), while both the integral form and a loosely associated extracellular (peripheral) form were observed during starvation. Formation and release of the latter rendered the starvation medium viscous. In addition, after 3 h of starvation in static conditions, less than 5% of the cells were motile, compared with 100% at the onset of starvation and approximately 80% subsequent to release of the peripheral EPS at 27 h of starvation. Inhibition of protein synthesis with chloramphenicol added before 3 h of starvation caused no increase in viscosity. However, addition of chloramphenicol at 3 h did not prevent the subsequent increase in viscosity displayed by S9 cells. The amount of integral EPS increased for both nontreated and chloramphenicol-treated S9 cells during the first hour of starvation, with a subsequent equal decrease. The chloramphenicol-treated cells, as well as cells of a transposon-generated mutant strain deficient in peripheral EPS formation, remained adhesive to a hydrophobic inanimate surface during the initial 5 h of starvation, whereas nontreated wild-type cells had progressively decreased adhesion capacity. During the initial 5 h of starvation, most of the nontreated cells but only a small fraction of the chloramphenicol-treated and mutant cells detached from the hydrophobic substratum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of cranberry on Escherichia coli cellular surface characteristics

The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.     

Efficacy of UV treatment in the management of bacterial adhesion on hard surfaces

The efficacy of UV treatment to control bacterial adhesion onto hard surfaces was investigated in laboratory conditions. The major characteristics necessary for biofilm formation like extracellular polymeric substance (EPS) production, carbohydrate and protein concentration in EPS, and adhesion ability onto hard surface were studied using two bacterial strains isolated from marine biofilms. The results showed that there was a considerable difference between the control and UV treated bacterial cultures in their viability, production of EPS, and adhesion ability. The protein and carbohydrate concentration of the EPS and the adhesion of bacterial cells to surface were also considerably reduced due to UV treatment. This study indicates that treatment of water with UV light may be used to control biofilm development on hard surfaces.     

Characteristics of the adhesive determinants of Lactobacillus fermentum 104

The adhesion of Lactobacillus fermentum 104-R and the variant strain 104-S to porcine gastric squamous epithelium was investigated. An epithelium-specific adhesion was detected for strain 104-S; however, strain 104-R expressed enhanced adhesion capacity to the control surfaces of polystyrene and bovine serum albumin. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. The adhesion pattern of bacterial cells in buffers of pH values ranging from 2 to 7 was determined. The adhesion of strain 104-S to epithelium was greater in a buffer with a higher pH value. On the other hand, adhesion of strain 104-R to the epithelium was rather unaffected by a change in pH. To the control surfaces of polystyrene or bovine serum albumin, the adhesion of both strains was greatest at pH 2 to 4. Treatment of strain 104-S with metaperiodate did not affect the adhesion to epithelium or polystyrene; however, protease treatment dramatically decreased the adhesion of both strains, thus suggesting that the determinants responsible for the adhesion were proteinaceous. Carbohydrates may be partially involved in the adhesion of 104-R because metaperiodate-treated cells adhered more poorly than control, iodate-treated cells. The adhesion-promoting components are most probably tightly bound to the cell wall, because washing with low-pH buffer (pH 1.2) or sodium dodecyl sulfate had no major effect on the adhesion.     

Characteristics of the adhesive determinants of Lactobacillus fermentum 104.

The adhesion of Lactobacillus fermentum 104-R and the variant strain 104-S to porcine gastric squamous epithelium was investigated. An epithelium-specific adhesion was detected for strain 104-S; however, strain 104-R expressed enhanced adhesion capacity to the control surfaces of polystyrene and bovine serum albumin. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. The adhesion pattern of bacterial cells in buffers of pH values ranging from 2 to 7 was determined. The adhesion of strain 104-S to epithelium was greater in a buffer with a higher pH value. On the other hand, adhesion of strain 104-R to the epithelium was rather unaffected by a change in pH. To the control surfaces of polystyrene or bovine serum albumin, the adhesion of both strains was greatest at pH 2 to 4. Treatment of strain 104-S with metaperiodate did not affect the adhesion to epithelium or polystyrene; however, protease treatment dramatically decreased the adhesion of both strains, thus suggesting that the determinants responsible for the adhesion were proteinaceous. Carbohydrates may be partially involved in the adhesion of 104-R because metaperiodate-treated cells adhered more poorly than control, iodate-treated cells. The adhesion-promoting components are most probably tightly bound to the cell wall, because washing with low-pH buffer (pH 1.2) or sodium dodecyl sulfate had no major effect on the adhesion.     

A high molecular mass constituent of cranberry juice inhibits helicobacter pylori adhesion to human gastric mucus

Because previous studies have shown that a high molecular mass constituent of cranberry juice inhibited adhesion of Escherichia coli to epithelial cells and coaggregation of oral bacteria, we have examined its effect on the adhesion of Helicobacter pylori to immobilized human mucus and to erythrocytes. We employed three strains of H. pylori all of which bound to the mucus and agglutinated human erythrocytes via a sialic acid-specific adhesin. The results showed that a high molecular mass constituent derived from cranberry juice inhibits the sialic acid-specific adhesion of H. pylori to human gastric mucus and to human erythrocytes.     

Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and Curli production on its resistance to chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log(10) CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 microg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12 degrees C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Extracellular polymeric substances responsible for bacterial adhesion onto solid surface

The influence of extracellular polymeric substances (EPS) on bacterial cell adhesion onto solid surfaces was investigated using 27 heterotrophic bacterial strains isolated from a wastewater treatment reactor. Cell adhesion onto glass beads was carried out by the packed-bed method and the results were discussed in terms of the amount of each EPS component produced and cell surface characteristics such as zeta potential and hydrophobicity. Protein and polysaccharides accounted for 75-89% of the EPS composition, indicating that they are the major EPS components. Among the polysaccharides, the amounts of hexose, hexosamine and ketose were relatively high in EPS-rich strains. For EPS-poor strains, the efficiency of cell adhesion onto glass beads increased as the absolute values of zeta potential decreased, suggesting that electrostatic interaction suppresses cell adhesion efficiency. On the other hand, the amounts of hexose and pentose exhibited good correlations with cell adhesiveness for EPS-rich strains, indicating that polymeric interaction due to the EPS covering on the cell surface promoted cell adhesion. It was concluded that, if the EPS amount is relatively small, cell adhesion onto solid surfaces is inhibited by electrostatic interaction, and if it is relatively large, cell adhesion is enhanced by polymeric interaction.     

Exopolysaccharide structure is not a determinant of host-plant specificity in nodulation of Vicia sativa roots

Exopolysaccharide (EPS)-deficient strains of the root nodule symbiote Rhizobium leguminosarum induce formation of abortive infection threads in Vicia sativa subsp. nigra roots. As a result, the nodule tissue remains uninfected. Formation of an infection thread can be restored by coinoculation of the EPS-deficient mutant with a Nod factor-deficient strain, which produces a similar EPS structure. This suggests that EPS contributes to host-plant specificity of nodulation. Here, a comparison was made of i) coinoculation with heterologous strains with different EPS structures, and ii) introduction of the pRL1JI Sym plasmid or a nod gene-encoding fragment in the same heterologous strains. Most strains not complementing in coinoculation experiments were able to nodulate V. sativa roots as transconjugants. Apparently, coinoculation is a delicate approach in which differences in root colonization ability or bacterial growth rate easily affect successful infection-thread formation. Obviously, lack of infection-thread formation in coinoculation studies is not solely determined by EPS structure. Transconjugation data show that different EPS structures can allow infection-thread formation and subsequent nodulation of V. sativa roots.     

Activity of the enzymes involved in the synthesis of exopolysaccharide precursors in an overproducing mutant ropy strain of Streptococcus thermophilus

The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus. These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production. The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain. The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors.     

The role of conditioning film formation in Pseudomonas aeruginosa PAO1 adhesion to inert surfaces in aquatic environments

Bacterial initial adhesion to inert surfaces in aquatic environments is highly dependent on the surface properties of the substratum, which can be altered significantly by the formation of conditioning films. In this study, the impact of conditioning films formed with extracellular polymeric substances (EPS) on bacterial adhesion was investigated. Adhesion of wild type Pseudomonas aeruginosa PAO1 to slides coated with model EPS components (alginate, humic substances, and bovine serum albumin (BSA)) was examined. Surface roughness of conditioning film coated slides was evaluated by atomic force microscopy (AFM), and its effect on the bacterial initial adhesion was not significant. X-ray photoelectron spectroscopy (XPS) studies were performed to determine the elemental surface compositions of bacterial cells and substrates. Results showed that bacterial adhesion to bare slides and slides coated with alginate and humic substances increased as ionic strength increased. Conversely, BSA coating enhanced bacterial adhesion at low ionic strength but hindered adhesion at higher ionic strength. It was concluded that forces other than hydrophobic and electrostatic interactions were involved in controlling bacterial adhesion to BSA coated surfaces. A steric model for polymer brushes that considers the combined influence of steric effects and DLVO interaction forces was shown to adequately describe the observed bacterial adhesion behaviors.     

Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log₁₀ CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 μg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12°C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Debaryomyces hansenii strains with different cell sizes and surface physicochemical properties adhere differently to a solid agarose surface

The initial adhesion of four Debaryomyces hansenii strains to a solid agarose surface was investigated and correlated with their cell size and some cell surface physicochemical properties, i.e. (i) hydrophobicity and (ii) electron donor/acceptor ability. One strain adhered very poorly, whereas the three other strains were more adhesive. The former strain had a very hydrophilic cell surface, whereas the latter strains had more hydrophobic cell surfaces. In addition, the strain with the lowest adhesion among the adhesive strains had a more hydrophobic cell surface than the two most adhesive strains. Finally, the more adhesive the strain was, the larger it was, and the better it was to donate electrons from its cell surface. These results show a clear relationship between the cell size, the cell surface physicochemical properties, and the initial adhesion of D. hansenii. A possible explanation of this relationship is discussed.     

Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence.

Pseudomonas solanacearum undergoes a spontaneous mutation that pleiotropically reduces extracellular polysaccharide (EPS) production, endoglucanase activity, and virulence and increases motility. We refer to the process that coordinately affects these traits as phenotype conversion (PC) and the resulting mutants as PC types. Previous research with the wild-type strain AW1 suggested that inactivation of a single locus could mimic phenotype conversion (T. P. Denny, F. W. Makini, and S. M. Brumbley, Mol. Plant-Microbe Interact. 1:215-223, 1988). Additional Tn5 mutagenesis of AW1 generated three more mutants (AW1-81, AW1-82, and AW1-84) that were indistinguishable from the PC type and one slightly leaky mutant (AW1-87); all four had single insertions in the same 4.0-kilobase (kb) EcoRI fragment that were responsible for the PC-like phenotype. Another insertion mutant, AW1-83, which lacks an insertion in this 4.0-kb fragment, resembled the PC type except that it was reversibly induced to produce wild-type levels of EPS when cultured adjacent to AW1. The wild-type region containing the gene that controls traits affected by phenotype conversion in AW1, designated phcA, was cloned on a 2.2-kb DNA fragment that restored all the phcA::Tn5 mutants and 11 independent spontaneous PC-type derivatives of AW1 to wild-type status. Homology with the phcA region was found in diverse wild-type strains of P. solanacearum, although restriction fragment length polymorphisms were seen. No major DNA alterations were observed in the phcA homologous region of PC types from strain AW1 or 82N. PC types from 7 of 11 conjugal strains of P. solanacearum were restored to EPS+ by phcA from AW1; however, only some PC types of strain K60 were restored, whereas others were not. We believe that a functional phcA gene is required to maintain the wild-type phenotype in P. solanacearum, and for most strains phenotype conversion results from a loss of phcA gene expression or the function of its gene product.     

Diversity of heteropolysaccharide-producing lactic acid bacterium strains and their biopolymers

Thirty-one lactic acid bacterial strains from different species were evaluated for exopolysaccharide (EPS) production in milk. Thermophilic strains produced more EPS than mesophilic ones, but EPS yields were generally low. Ropiness or capsular polysaccharide formation was strain dependent. Six strains produced high-molecular-mass EPS. Polymers were classified into nine groups on the basis of their monomer composition. EPS from Enterococcus strains were isolated and characterized.     

Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics

Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as Streptococcus thermophilus and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium. In addition, the acidification rates of the different strains were compared with their milk clotting behaviors. The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening. EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium. Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium. An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa. A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa). Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished. Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method. All of the EPS from the S. thermophilus strains tested were classified into six groups according to their monomer compositions. Apart from galactose and glucose, other monomers, such as (N-acetyl)galactosamine, (N-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents. Three strains were found to produce EPS containing (N-acetyl)glucosamine, which to our knowledge was never found before in an EPS from S. thermophilus. Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures. Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures.