Retention of bacteria on a substratum surface with micro-patterned hydrophobicity

Bacteria adhere to almost any surface, despite continuing arguments about the importance of physico-chemical properties of substratum surfaces, such as hydrophobicity and charge in biofilm formation. Nevertheless, in vivo biofilm formation on teeth and also on voice prostheses in laryngectomized patients is less on hydrophobic than on hydrophilic surfaces. With the aid of micro-patterned surfaces consisting of 10-microm wide hydrophobic lines separated by 20-microm wide hydrophilic spacings, we demonstrate here, for the first time in one and the same experiment, that bacteria do not have a strong preference for adhesion to hydrophobic or hydrophilic surfaces. Upon challenging the adhering bacteria, after deposition in a parallel plate flow chamber, with a high detachment force, however, bacteria were easily wiped-off hydrophobic lines, most notably when these lines were oriented parallel to the direction of flow. Adhering bacteria detached slightly less from the hydrophilic spacings in between, but preferentially accumulated adhering on the hydrophilic regions close to the interface between the hydrophilic spacings and hydrophobic lines. It is concluded that substratum hydrophobicity is a major determinant of bacterial retention while it hardly influences bacterial adhesion.     

Surface properties of Staphylococcus aureus affecting chemiluminescence response of human phagocytes

To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.     

Adhesion of coagulase-negative staphylococci to biomaterials

The adhesion of two Staphylococcus epidermidis strains and one Staphylococcus saprophyticus strain on to poly(tetrafluorethylene-co-hexafluorpropylene) (FEP)-fluorocarbon and cellulose acetate was studied in vitro. Both S. epidermidis strains showed a more hydrophobic character than the encapsulated S. saprophyticus as determined by the bacterial affinity towards xylene. Staphylococcus epidermidis showed a significantly higher adhesion on to the hydrophobic FEP than S. saprophyticus. The adhesion of staphylococci on to the more hydrophilic cellulose acetate was always low. Treatment of S. epidermidis with pepsin or extraction with aqueous phenol yielded cells with a decreased hydrophobicity, which resulted in a decreased adhesion on to FEP. Cells with a decreased hydrophobicity showed a lower rate of reaggregation in suspension. The hydrophobicity and the adhesion on the FEP of S. epidermidis were not affected by exposure to a subminimal inhibitory concentration of penicillin. The strong interaction between S. epidermidis and FEP, which appeared not to be influenced by the age or the metabolic stage of the bacteria, is mainly caused by hydrophobic bonding.     

Surface properties of lactobacilli isolated from the small intestine of pigs

One hundred wild-type strains of the genus Lactobacillus were isolated from the small intestine of newly-slaughtered pigs up to 6 months of age. Cell surface hydrophobicity and capsule formation were studied on a number of strains. Strains showing high surface hydrophobicity as measured by the salt-aggregation test and hydrophobic interaction chromatography on Octyl Sepharose were commonly found to adhere in high numbers to isolated pig intestinal epithelial cells. Heat and protease treatment of bacteria of high surface hydrophobicity, including autoaggregating strains in phosphate-buffered saline, showed a drastic decline in this surface property. Three hydrophilic strains (LBp 1044, 1068 and 1073) also showed binding to intestinal cells but at a lower level (approx. 5 bacteria/cell) as compared with the best binding hydrophobic strain (LBp 1063, approx. 11 bacteria/cell). These findings suggest that different or multiple adhesion mechanisms may be involved in the colonization of the small intestinal mucosa of pigs. Cultures of selected strains grown in liquid media rich in carbohydrates did not affect their hydrophobic cell surface character. Therefore it seems less likely that carbohydrate capsule polymers are the major determinants of intestinal colonization of lactobacilli in pigs.     

Surface properties of lactobacilli isolated from the small intestine of pigs

One hundred wild-type strains of the genus Lactobacillus were isolated from the small intestine of newly-slaughtered pigs up to 6 months of age. Cell surface hydrophobicity and capsule formation were studied on a number of strains. Strains showing high surface hydrophobicity as measured by the salt-aggregation test and hydrophobic interaction chromatography on Octyl Sepharose were commonly found to adhere in high numbers to isolated pig intestinal epithelial cells. Heat and protease treatment of bacteria of high surface hydrophobicity, including autoaggregating strains in phosphate-buffered saline, showed a drastic decline in this surface property. Three hydrophilic strains (LBp 1044, 1068 and 1073) also showed binding to intestinal cells but at a lower level (approx. 5 bacteria/cell) as compared with the best binding hydrophobic strain (LBp 1063, approx. 11 bacteria/cell). These findings suggest that different or multiple adhesion mechanisms may be involved in the colonization of the small intestinal mucosa of pigs. Cultures of selected strains grown in liquid media rich in carbohydrates did not affect their hydrophobic cell surface character. Therefore it seems less likely that carbohydrate capsule polymers are the major determinants of intestinal colonization of lactobacilli in pigs.     

Hydrophobicity, Adhesion, and Surface-Exposed Proteins of Gliding Bacteria

The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by an assay of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure.     

Determination of bacterial cell surface hydrophobicity of single cells in cultures and in wastewater in situ

Bacterial cell surface hydrophobicity is one of the most important factors that influence bacterial adhesion. A new method, microsphere adhesion to cells, for measuring bacterial cell surface hydrophobicity was developed. Microsphere adhesion to cells is based on microscopic enumeration of hydrophobic, fluorescent microspheres attaching to the bacterial surface. Cell surface hydrophobicity estimated by microsphere adhesion to cells correlates well with adhesion of bacteria to hydrocarbons or hydrophobic interaction chromatography for a set of hydrophilic and hydrophobic bacteria (linear correlation coefficients, R², were 0.845 and 0.981 respectively). We also used microsphere adhesion to cells to investigate the in situ properties of individual free-living bacteria directly in activated sludge. Results showed that the majority of the bacteria were hydrophilic, indicating the importance of cell surface hydrophobicity for bacterial adhesion in sludge, and for the overall success of the wastewater treatment process.     

Role of hydrophobicity in bacterial adherence to carbon nanostructures and biofilm formation

The role of cell and surface hydrophobicity in the adherence of the waterborne bacterium Mycobacterium smegmatis to nanostructures and biofilm formation was investigated. Carbon nanostructures (CNs) were synthesized using a flame reactor and deposited on stainless steel grids and foils, and on silicon wafers that had different initial surface hydrophobicities. Surface hydrophobicity was measured as the contact angle of water droplets. The surfaces were incubated in suspensions of isogenic hydrophobic and hydrophilic strains of M. smegmatis and temporal measurements of the numbers of adherent cells were made. The hydrophobic, rough mutant of M. smegmatis adhered more readily and formed denser biofilms on all surfaces compared to its hydrophilic, smooth parent. Biofilm formation led to alterations in the hydrophobicity of the substratum surfaces, demonstrating that bacterial cells attached to CNs are capable of modifying the surface characteristics.     

Compositional factors influencing cell surface hydrophobicity ofPasteurella multocida variants

The influence of macromolecules other than lipopolysaccharide on the hydrophobic properties ofPasteurella multocida was investigated by assessing cell surface hydrophobicity (CSH) after experimentally modifying surfaces of various strains. CSH of hydrophobic variants was enhanced by growth on blood-supplemented medium and mechanical shearing, whereas chloramphenicol, oxytetracycline, trypsin, and pronase E treatments decreased CSH. No such modifications were observed for hydrophilic strains. Microscopic observations revealed hydrophilic strains to be heavily encapsulated in contrast to hydrophobic strains. Repeated subculturing reduced encapsulation with a concomitant increase in CSH for one hydrophilic strain while exerting no changes in the other hydrophilic strain examined. Hyaluronidase removal of capsular material from a serotype A strain resulted in increased CSH; subsequent exposure to pronase E resulted in partial restoration of hydrophilicity. These data suggest the encapsulation of hydrophilicP. multocida strains masks a relatively hydrophobic surface that is conferred, at least in part, by the presence of one or more surface-exposed proteins common to both hydrophilic and hydrophobic variants.     

Influence of the Gas-Water Interface on Transport of Microorganisms through Unsaturated Porous Media

In this article, a new mechanism influencing the transport of microorganisms through unsaturated porous media is examined, and a new method for directly visualizing bacterial behavior within a porous medium under controlled chemical and flow conditions is introduced. Resting cells of hydrophilic and relatively hydrophobic bacterial strains isolated from groundwater were used as model microorganisms. The degree of hydrophobicity was determined by contact-angle measurements. Glass micromodels allowed the direct observation of bacterial behavior on a pore scale, and three types of sand columns with different gas saturations provided quantitative measurements of the observed phenomena on a porous medium scale. The reproducibility of each break-through curve was established in three to five repeated experiments. The data collected from the column experiments can be explained by phenomena directly observed in the micromodel experiments. The retention rate of bacteria is proportional to the gas saturation in porous media because of the preferential sorption of bacteria onto the gas-water interface over the solid-water interface. The degree of sorption is controlled mainly by cell surface hydrophobicity under the simulated groundwater conditions because of hydrophobic forces between the organisms and the interfaces. The sorption onto the gas-water interface is essentially irreversible because of capillary forces. This preferential and irreversible sorption at the gas-water interface strongly influences the movement and spatial distribution of microorganisms.     

Effect of Substrate and Cell Surface Hydrophobicity on Phosphate Utilization in Bacteria

We measured the rates of utilization of hydrophobic and hydrophilic phosphate compounds in gram-negative bacteria with different surface hydrophobicities, isolated from wetland habitats. Three hydrophobic and two hydrophilic bacterial species were selected for study by measuring cell adherence to hydrocarbons. The bacteria were grown under phosphorus-limited conditions with P(infi), hydrophilic (beta)-glycerophosphate, or hydrophobic phosphatidic acid as the phosphate source. Hydrophilic bacteria grew most rapidly on P(infi), followed by (beta)-glycerophosphate. Phosphatidic acid did not support growth or did so at a much later time (40 h) than did the other phosphate treatments. Although all hydrophobic species grew well on these substrates, the rate of growth of two Acinetobacter baumannii isolates on phosphatidic acid exceeded the rate of growth on phosphate or (beta)-glycerophosphate. A membrane phospholipid and lipopolysaccharide were used as a source of phosphorus by hydrophobic species, whereas hydrophilic species could not use the membrane phospholipids and used lipopolysaccharide to a lesser extent. Besides hydrophobic interaction between cells and substrate, phosphatase activity, which was cell bound in hydrophilic species but 30 to 50% unbound in hydrophobic species, affected cell growth. Dialyzed culture supernatant containing phosphatase from hydrophobic species increased the phosphate availability to hydrophilic species. Additionally, cellular extracts from a hydrophilic species, when added to hydrophilic cells, permitted growth on hydrophobic phosphate sources. Naturally occurring amphiphilic humic acids affected the utilization of P(infi) and (beta)-glycerophosphate in bacteria with hydrophilic surfaces but did not affect hydrophobic bacteria. Our results indicate that hydrophobic phosphate sources can be used by bacteria isolated from aquatic environments as the sole phosphorus source for growth. This utilization, in part, appears to be related to cell surface hydrophobicity and extracellular enzyme production.     

How pathogenic bacteria evade mammalian sabotage in the battle for iron

Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for bacteria growing in animals because of its hydrophobicity and its sequestration by the mammalian protein siderocalin, a component of the innate immune system. However, pathogenic strains of Escherichia coli and Salmonella use enzymes encoded by the iroA gene cluster to tailor enterobactin by glycosylation and linearization. The resulting modified forms of enterobactin, known as salmochelins, can evade siderocalin and are less hydrophobic than enterobactin, restoring this siderophore's iron-scavenging ability in mammals.     

The role of bacterial cell wall hydrophobicity in adhesion

In this study, the adhesion of bacteria differing in surface hydrophobicity was investigated. Cell wall hydrophobicity was measured as the contact angle of water on a bacterial layer collected on a microfilter. The contact angles ranged from 15 to 70 degrees. This method was compared with procedures based upon adhesion to hexadecane and with the partition of cells in a polyethylene glycol-dextran two-phase system. The results obtained with these three methods agreed reasonably well. The adhesion of 16 bacterial strains was measured on sulfated polystyrene as the solid phase. These experiments showed that hydrophobic cells adhered to a greater extent than hydrophilic cells. The extent of adhesion correlated well with the measured contact angles (linear regression coefficient, 0.8).     

Low cell surface hydrophobicity is one of the key factors for high butanol tolerance of Lactic acid bacteria

Highly butanol‐tolerant strains have always been attractive because of their potential as microbial hosts for butanol production. However, due to the amphiphilic nature of 1‐butanol as a solvent, the relationship between the cell surface hydrophobicity and butanol resistance remained ambiguous to date. In this work, the quantitatively estimated cell surface hydrophobicity of 74 Lactic acid bacteria strains were juxtaposed to their tolerance to various butanol concentrations. The obtained results revealed that the strains’ hydrophobicity was inversely proportional to their butanol tolerance. All highly butanol‐resistant strains were hydrophilic (cell surface hydrophobicity<1%), whereas the more hydrophobic the strains were, the more sensitive to butanol they were. Furthermore, cultivation at increasing butanol concentrations showed a clear tendency to decrease the level of hydrophobicity in all tested organisms, thus suggesting possible adaptation mechanisms. Purposeful reduction of cell surface hydrophobicity (by removal of S‐layer proteins from the cell envelope) also led to an increase of butanol resistance. Since the results covered 23 different Lactic acid bacteria species of seven genera, it could be concluded that regardless of the species, the lower degree of cells’ hydrophobicity clearly correlates with the higher level of butanol tolerance.     

The role of bacterial cell wall hydrophobicity in adhesion.

In this study, the adhesion of bacteria differing in surface hydrophobicity was investigated. Cell wall hydrophobicity was measured as the contact angle of water on a bacterial layer collected on a microfilter. The contact angles ranged from 15 to 70 degrees. This method was compared with procedures based upon adhesion to hexadecane and with the partition of cells in a polyethylene glycol-dextran two-phase system. The results obtained with these three methods agreed reasonably well. The adhesion of 16 bacterial strains was measured on sulfated polystyrene as the solid phase. These experiments showed that hydrophobic cells adhered to a greater extent than hydrophilic cells. The extent of adhesion correlated well with the measured contact angles (linear regression coefficient, 0.8).     

Autoaggregation,hydrophobic, and hydrophylic properties of Moraxella catarrhalis strains

The hydrophobicity of the bacterial cell surface was evaluated via the salt aggregation test (SAT) in 58 strains (19 from the lower and 39 from the upper respiratory tracts) of Moraxella catarrhalis in hospitalized patients aged 25 to 65. Based on the SAT results, the strains were divided into three groups: autoaggregating (highly hydrophobic), hydrophobic, and hydrophilic. At a temperature of 37 degrees C, the autoaggregating, hydrophobic or hydrophilic properties did not depend on the choice of a medium, whereas at 22 degrees C the investigated properties did (p<0.0001). Taking into account the origin of the strains (lower vs. upper respiratory tract), it was found that: in the strains cultivated in liquid medium, both highly hydrophobic, hydrophobic and hydrophilic surfaces were present with a comparative frequency, independent of the strain isolation site and cultivation conditions; strains with highly hydrophobic and hydrophobic surfaces, but only those cultivated on solid media at 22 degrees C, were much more often isolated from sputum rather than from nose and throat swabs, whereas a statistically significant incidence of hydrophilic strains was found in samples from the upper rather than lower respiratory tract.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Role of hydrophobic surface proteins in mediating adherence of group B streptococci to epithelial cells.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.     

Correlation between the activities of alpha-helical antimicrobial peptides and hydrophobicities represented as RP HPLC retention times

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.