Measurement of the kinetics of bacterial adherence to hexadecane in polystyrene cuvettes

Abstract The kinetics of bacterial adherence to hexadecane were measured using disposable polystyrene cuvettes. The rate of adherence was exponential, and was itself linearly dependent on the water:hexadecane ratio employed. The dependency of the rate of adherence on the water:hexadecane ratio, termed the removal coefficient, varied from 4.7 min⁻¹ for Streptococcus pyogenes to 72 min⁻¹ for Acinetobacter calcoaceticus . The removal coefficient of Serratia marcescens was a function of growth temperature, 48 min⁻¹ following growth at 30°C as opposed to 5.8 min⁻¹ for 35°C-grown cells.     

Concentration of larvicidal Bacillus spores at the water surface by adherence to oil droplets

Adherence of two larvicidal spores, Bacillus sphaericus 2297 and Bacillus thuringiensis israelensis, to hexadecane and corn oil was determined. Adherence was greatly promoted in the presence of 2 M ammonium sulphate, reaching levels of up to 89%. The data suggest that larvicidal spores may be concentrated at their operational niche (the water surface) by flotation, adhering to the surface of oil droplets. The data suggest that adherence of the spores to oil droplets may enable their concentration at the water surface, where they can effectively kill mosquito larvae.     

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Hydrophobic interactions within biofilms of nitrifying and denitrifying bacteria in biofilters

Hydrophobicity of the solid surface and microbial cell surface is important factor for the development of biofilms applied in bioengineering systems. An adsorption of phenanthrene was used for analysis of the hydrophobicity of support fibers and bacterial cell surfaces within the biofilter of wastewater. The adsorption of phenanthrene was measured by synchronous fluorescence spectrometry. Cell surface hydrophobicity does not depend on the fixation procedure, pH of microbial suspension, and has no clear correlation with an adherence of the cells to hexadecane droplets. Notwithstanding high hydrophobicity of bacterial cells, the hydrophobicity of intact biofilm is determined by the hydrophobicity of the support fibers. New indexes were proposed to evaluate the reactor performance related with hydrophobic interactions within the biofilm. These indexes showed that significant share of hydrophobic sites within the nitrifying biofilm is protected from the hydrophobic interactions between the cells and environment.     

Adherence of emulsan-producing Acinetobacter calcoaceticus to hydrophobic liquids

Summary The adherence of Acinetobacter calcoaceticus ATCC 31012 cells to hexadecane and perfluorocarbon FC-43 was measured using the Bacterial Adherence To Hydrocarbon (BATH) assay. In batch culture the adherence of cells to both hydrophobic liquids increased sharply during the exponential growth phase and remained high for the remainder of the culture period. No correlation was found between the surface emulsan concentration and the adherence to perfluorocarbon FC-43 and hexadecane. In continuous cultures, the production of cell-free emulsan was found to be growth-associated. The adherence to both hydrophobic liquids decreased with increasing dilution rate while the amount of surface emulsan increased. Furthermore, exogenously added emulsan decreased the adherence to hydrophobic liquids. Thus, the accumulation of surface emulsan does not appear to have a beneficial effect for cell adherence to hydrophobic liquids.     

Trypsin-susceptible cell surface characteristics of Streptococcus sanguis

The adherence of Streptococcus sanguis to saliva-coated hydroxylapatite was markedly reduced by treatment of the cells with trypsin. In Scatchard plots of adherence data, protease-treated S. sanguis did not exhibit the characteristic positive slopes, suggesting that trypsin prevented cooperative interactions between the cells and artificial pellicle. Trypsin also reduced the tendency of S. sanguis to bind to hexadecane and to octyl-Sepharose. When sodium dodecyl sulfate was used to elute S. sanguis from columns of octyl-Sepharose, it was observed that the elution profiles of trypsin-treated cells were more complex than those of control cells. Water and salts were incapable of removing the cells from octyl-Sepharose. The results suggest that adherence to saliva-coated hydroxylapatite, binding to hexadecane and to octyl-Sepharose depend on trypsin-susceptible cell surface molecules.     

Separation of amphipathic proteins based on adsorption to hexadecane:water interfaces

A method is proposed for the relatively simple and rapid separation of amphipathic biopolymers, based on adsorption onto and desorption from the surface of hexadecane droplets. Adsorption to the hexadecane:water interface was carried out by mixing hexadecane with aqueous protein solutions at room temperature. Desorption was performed by consecutive solidification and melting of the liquid hydrocarbon (m.p. 18 degrees C), resulting in coalescence of the droplets and reappearance of the desorbed moiety in the bulk aqueous phase. Of interest was the observation that lysozyme remains enzymatically active following this procedure.     

Putative role of a 70 kDa outer-surface protein in promoting cell-surface hydrophobicity of Serratia marcescens RZ

Serratia marcescens RZ has been previously shown to possess pronounced cell-surface hydrophobicity, as evidenced by its affinity for hydrocarbons and polystyrene. The present report suggests the involvement of a 70 kDa protein, serraphobin, in this phenomenon. The 70 kDa protein was recovered from both the cell surface and culture supernatant of hydrophobic wild-type cells, but was either totally absent or present in minor quantities in hydrophobicity-deficient mutants. Similarly, loss of hydrophobicity of RZ cells following growth at 39 degrees C was accompanied by loss of the protein. Serraphobin was capable of binding to hexadecane droplets following a brief mixing procedure, and could be desorbed by solidifying and melting the hexadecane phase.     

Effect of Cetylpyridinium Chloride on Microbial Adhesion to Hexadecane and Polystyrene

Microbial adhesion at the oil-water interface is a subject of both basic interest (e.g., as a technique for the measurement of hydrophobicity) and applied interest (e.g., for use in two-phase oil-water mouthwashes for the desorption of oral microorganisms). In general, surfactants inhibit microbial adhesion to oils and other hydrophobic surfaces. In the present study, we demonstrated that the cationic surfactant cetylpyridinium chloride (CPC) significantly enhanced microbial adhesion to hexadecane and various oils, as well as to the solid hydrophobic surface polystyrene. CPC increased adhesion to hexadecane of Escherichia coli, Candida albicans and Acinetobacter calcoaceticus MR-481 and of expectorated oral bacteria from near 0% to over 90%. The CPC concentration required for optimal enhancement of adhesion was a function of the initial cell density. This phenomenon was inhibited by high salt concentrations and, in the case of E. coli, by a low pH. CPC-pretreated cells were able to bind to hexadecane, but CPC-pretreated hexadecane was unable to bind untreated cells. Another cationic, surface-active antimicrobial agent, chlorhexidine gluconate, was similarly able to promote microbial adhesion to hexadecane. The results suggest that (i) CPC enhances microbial adhesion to hexadecane by binding via electrostatic interactions at the cell surface, thus diminishing surface charge and increasing cell surface hydrophobicity, and (ii) this phenomenon may have applications in oral formulations and in the use of hydrocarbon droplets as a support for cell immobilization.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Virulent aggregates of Streptococcus pyogenes are generated by homophilic protein-protein interactions

Many strains of the important human pathogen Streptococcus pyogenes form aggregates when grown in vitro in liquid medium. The present studies demonstrate that this property is crucial for the adherence, the resistance to phagocytosis and the virulence of S. pyogenes. A conserved sequence of 19 amino acid residues (designated AHP) was identified in surface proteins of common S. pyogenes serotypes. This sequence was found to promote bacterial aggregation through homophilic protein-protein interactions between AHP-containing surface proteins of neighbouring bacteria. A synthetic AHP peptide inhibited S. pyogenes aggregation, reduced the survival of S. pyogenes in human blood and attenuated its virulence in mice. In contrast, mutant bacteria devoid of surface proteins containing AHP-related sequences did not aggregate or adhere to epithelial cells. These bacteria are also rapidly killed in human blood and show reduced virulence in mice, underlining the pathogenic significance of the AHP sequence and S. pyogenes aggregation.     

Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.     

Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable to both light and electron microscopic observation.     

Role of adherence in growth of Acinetobacter calcoaceticus RAG-1 on hexadecane.

The high affinity of Acinetobacter calcoaceticus RAG-1 for liquid hydrocarbons permitted the isolation of a spontaneous nonadherent mutant, MR-481. Strain MR-481 exhibited no significant affinity for three test hydrocarbons, yet resembled the wild type in many properties, including production of the extracellular emulsifying agent emulsan. To study the role of adherence in growth on hydrocarbons, RAG-1 and MR-481 were compared for growth on hexadecane under conditions of limited agitation and at low initial cell densities. Adherent RAG-1 cells were able to grow rapidly under these conditions, whereas nonadherent MR-481 cells failed to grow for at least 54 h. However, the addition of emulsan either initially or at various times after inoculation enabled the nonadherent MR-481 cells to grow on hexadecane. Growth was not the result of reversion of MR-481 from nonadherent to adherent cells. The data demonstrate that adherence is a crucial factor in the growth of A. calcoaceticus RAG-1 on hexadecane in the absence of extracellular emulsification of the substrate.     

DNA from Serratia marcescens confers a hydrophobic character in Escherichia coli

Abstract In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli , E. coli DH5α cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized components) on the transformant outer surface.     

Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers.

Polycationic polymers have been noted for their effects in promoting cell adhesion to various surfaces, but previous studies have failed to describe a mechanism dealing with this type of adhesion. In the present study, three polycationic polymers (chitosan, poly-L-lysine, and lysozyme) were tested for their effects on microbial hydrophobicity, as determined by adhesion to hydrocarbon and polystyrene. Test strains (Escherichia coli, Candida albicans, and a nonhydrophobic mutant, MR-481, derived from Acinetobacter calcoaceticus RAG-1) were vortexed with hexadecane in the presence of the various polycations, and the extent of adhesion was measured turbidimetrically. Adhesion of all three test strains rose from near zero values to over 90% in the presence of low concentrations of chitosan (125 to 250 micrograms/ml). Adhesion occurred by adsorption of chitosan directly to the cell surface, since E. coli cells preincubated in the presence of the polymer were highly adherent, whereas hexadecane droplets pretreated with chitosan were subsequently unable to bind untreated cells. Inorganic cations (Na+, Mg2+) inhibited the chitosan-mediated adhesion of E. coli to hexadecane, presumably by interfering with the electrostatic interactions responsible for adsorption of the polymer to the bacterial surface. Chitosan similarly promoted E. coli adhesion to polystyrene at concentrations slightly higher than those which mediated adhesion to hexadecane. Poly-L-lysine also promoted microbial adhesion to hexadecane, although at concentrations somewhat higher than those observed for chitosan. In order to study the effect of the cationic protein lysozyme, adhesion was studied at 0 degree C (to prevent enzymatic activity), using n-octane as the test hydrocarbon. Adhesion of E. coli increased by 70% in the presence of 80 micrograms of lysozyme per ml. When the negatively charged carboxylate residues on the E. coli cell surface were substituted for positively charged ammonium groups, the resulting cells became highly hydrophobic, even in the absence of polycations. The observed "hydrophobicity" of the microbial cells in the presence of polycations is thus probably due to a loss of surface electronegativity. The data suggest that enhancement of hydrophobicity by polycationic polymers is a general phenomenon.     

Two distinct pathways for the invasion of Streptococcus pyogenes in non-phagocytic cells

Adherence to and invasion of epithelial cells represent important pathogenic mechanisms of Streptococcus pyogenes . A fibronectin-binding surface protein of S. pyogenes , SfbI protein, has been implicated in both adherence and invasion processes. Invasion of SfbI-containing strains has been suspected to be responsible for the failure of antibiotics treatment to eradicate S. pyogenes . In this study, we tested the adherence and invasion properties of two well-characterized clinical isolates: A40, which expresses SfbI; and A8, which is SfbI negative and is unable to bind fibronectin. In strain A40, SfbI was the main factor required for attachment and invasion by using fibronectin as a bridging molecule and the α₅β₁ integrin as cellular receptor. The uptake process was characterized by the generation of large membrane invaginations at the bacteria–cell interface without evidence of actin recruitment or cellular injury. A40 cells were located in phagosomes and, only 24 h after infection, a consistent part of the bacterial population reached the cytoplasm. In contrast, uptake of strain A8 required major rearrangements of cytoskeletal proteins underneath attached bacteria. In A8, a proteinaceous moiety was involved, which does not interact with α₅β₁ or need any known bridging molecule. Bacterial attachment stimulated elongation and massive recruitment of neighbouring microvilli, which fused to surround streptococcal chains. They led to the generation of large pseudopod-like structures, which engulfed bacteria that were rapidly released and replicated in the cytoplasm. The identification of two completely different uptake pathways reported here provided further evidence regarding the diversity of S. pyogenes isolates and might contribute towards understanding the pathogenesis and persistence of S. pyogenes .