Reversal of the surface effects of chlorhexidine diacetate on cells of Providencia stuartii

Chlorhexidine diacetate (CHA) increased the hydrophobicity of the cell surface of cells of three strains of Providencia stuartii. Removal of at least some of the CHA from the cells by washing them with an appropriate antidote partially reversed the hydrophobicity-increasing action of the biguanide. The effects of other treatments on cell surface hydrophobicity were examined with these strains and, for comparison, with two strains each of Escherichia coli and Pseudomonas aeruginosa: ethyl-enediamine tetraacetic acid affected all strains, although not to the same extent, whereas thermal injury (55°C) produced marked changes only with the two E. coli strains.     

Effect of trace elements on surface hydrophobicity and adherence of Escherichia coli to uroepithelial cells

Trace elements have significant effect on the physiology of bacteria. Variation in the concentration of trace elements may affect the expression of virulence by microorganisms. The effect of trace elements on hydrophobicity and adherence of E.coli to uroepithelial cells was studied. Increasing concentrations of Ca2+, Mg2+, Fe3+ and Zn2+ significantly decreased the surface hydrophobicity. Toxic trace elements like Co2+, Cu2+, Mn2+ and Ni2+ did not alter surface hydrophobicity. With regards to adherence of E.coli to uroepithelial cells, only Mg2+ had significant effect. Toxic trace elements decreased the rate of cell adherence. The pathogenic strains of E.coli showed higher surface hydrophobicity and better cell adherence compared to the nonpathogenic strains. There was good correlation between surface hydrophobicity and cell adherence at higher concentrations (0.1 to 0.2mM) of Fe2+ and Zn2+. The results indicated that trace elements can significantly affect surface hydrophobicity and adherence of E.coli to uroepithelial cells. Such effect may have a significant impact on the initial stages of bacterial infection.     

Influence of surface cell structures on physicochemical properties of Escherichia coli

The partition of cells in a polyethylene glycol-dextran two phase system was used to compare the relative hydrophobicity of E. coli strains expressing different surface structures. The role of fimbriae and surface antigens on the behavior partition was investigated. The strains expressing PAP fimbriae and/or O-antigen showed a higher surface hydrophobicity than strains which express only type 1 fimbriae and/or R-antigen. No relation was found between K and H antigen and hydrophobicity measurements. The influence of surface structures on electrophoretic mobility has been evaluated. The polysaccharide capsules of AL 213 and AL 499 strains generated a high EPM. For non capsulated E. coli the EPM of rough strains (AL 46, 382) is higher than smooth strain (AL52).     

Fimbriation, surface hydrophobicity and serum resistance in uropathogenic strains of Escherichia coli

Abstract A total of 80 Escherichia coli strains were examined for expression of P-fimbriae, mannose-sensitive haemagglutination (MSHA) and mannose-resistant haemagglutination (MRHA) of human group A erythrocytes and guinea pig erthrocytes, cell surface hydrophobicity and resistance to serum bactericidal activity. Isolates were obtained from urine of children and adults, either with acute pyelonephritis ( n = 15 and n = 12) or lower urinary tract infection (UTI) ( n = 30 and n = 23, respectively). Results obtained showed that, in E. coli strains isolated both from children and adults with lower UTI, significant differences were not found concerning the incidence of P-fimbriae, cell surface hydrophobicity and serum resistance. In pyelonephritogenic E. coli isolated from children and adults, the incidence of P-fimbriae and cell surface hydrophobicity was associated more frequently with the former (87% vs. 42% and 100% vs. 67%, P < 0.05), while serum resistance was associated with the latter (47% vs. 67%, P < 0.05).     

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.     

Effect of chlorhexidine and phenoxyethanol on cell surface hydrophobicity of Gram-positive and Gram-negative bacteria

The surfaces of mutants of Escherichia coli and Pseudomonas aeruginosa were markedly more hydrophobic than the corresponding wild types, as were the latter when the organisms were pre-treated with chlorhexidine diacetate (CHA) or phenoxyethanol (POE). A combination of CHA and POE demonstrated that only at higher concentrations was there a marked effect on hydrophobicity compared with that of either drug used alone. The three methods used to determine hydrophobicity correlated well as long as constant conditions were employed.     

The effects of three non-antibiotic, antimicrobial agents on the surface hydrophobicity of certain micro-organisms evaluated by different methods

The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.     

Escherichia coli strains of serogroup O139 isolated from oedema disease of swine bind fibronectin

Abstract 15 Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with ¹²⁵I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding than Staphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7 E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding than S. aureus Cowan 1 cells. E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells of S. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.     

Mechanism of resistance of Bacillus subtilis spores to chlorhexidine

Chlorhexidine diacetate (CHA) was rather more sporicidal at 20C to ureadithreitol-sodium lauryl sulphate (UDS)-treated spores of Bacillus subtilis NCTC 8236 than to urea-dithiothreitol (UDT)-treated or normal (untreated) spores. UDS spores adsorbed more CHA from solution than did the other two forms. No differences in hydrophobicity, as determined by hydrophobic interaction chromatography (HIC) or bacterial adherence to hydrocarbon (BATH), could be detected between the three spore types. Germinating spores took up much less CHA than did outgrowing spores. Germinating cells were considerably more hydrophobic, as measured by the BATH technique, than outgrowing cells or normal spores. Chlorhexidine diacetate increased the apparent hydrophobicity of the two latter forms, but this effect could be partially reversed by subsequent exposure to a non-ionic surfactant.     

Mechanism of resistance of Bacillus subtilis spores to chlorhexidine

Chlorhexidine diacetate (CHA) was rather more sporicidal at 20 degrees C to urea-dithreitol-sodium lauryl sulphate (UDS)-treated spores of Bacillus subtilis NCTC 8236 than to urea-dithiothreitol (UDT)-treated or normal (untreated) spores. UDS spores adsorbed more CHA from solution than did the other two forms. No differences in hydrophobicity, as determined by hydrophobic interaction chromatography (HIC) or bacterial adherence to hydrocarbon (BATH), could be detected between the three spore types. Germinating spores took up much less CHA than did outgrowing spores. Germinating cells were considerably more hydrophobic, as measured by the BATH technique, than outgrowing cells or normal spores. Chlorhexidine diacetate increased the apparent hydrophobicity of the two latter forms, but this effect could be partially reversed by subsequent exposure to a non-ionic surfactant.     

Hydrophobic properties of Providencia stuartii and other Gram-negative bacteria measured by hydrophobic interaction chromatography

A hydrophobic interaction chromatography (HIC) procedure was used to compare the relative surface hydrophobicity of three Providencia stuartii strains and wild type and envelope mutants of Escherichia coli and Pseudomonas aeruginosa. Providencia stuartii strain Pv2 adsorbed to a greater extent to octyl- and phenyl-Sepharose than did Pv67. The HIC technique showed a significant difference in surface hydrophobicity between wild type and envelope mutant strains, the latter being considerably more hydrophobic. Pre-treatment of cell suspensions with chlorhexidine produced further changes in the hydrophobic nature of all the strains. Moreover, HIC provides a convenient and rapid alternative means of screening strains for a property potentially associated with adhesiveness.     

Fibronectin binding and cell-surface hydrophobicity of attaching effacing enteropathogenic Escherichia coli strains isolated from newborn and weanling rabbits with diarrhoea

Abstract 32 different strains of Escherichia coli isolated from rabbits with diarrhoea were studied for cell-surface properties which may be involved in intestinal colonisation. Strains isolated from diarrhoeic suckling (6 strains) and weaning (26 strains) rabbits which were shown to attach to brush borders in vivo, showed high relative cell-surface hydrophobicity as determined by the Salt Aggregation Test (SAT) when grown on Colonisation Factor Antigen (CFA) agar at 33°C. Cells of these strains grown to express surface hydrophobicity were also defined as high, moderate or low binders of ¹²⁵I-fibronectin or its ¹²⁵I-29-kDa fragment in a standard binding assay. Based on these findings, we propose that binding to intestinal cell surface (mucus)-associated fibronectin may be an early important step in intestinal colonisation of the small bowel in enteropathogenic E. coli (EPEC) diarrhoea in rabbits and other animal species.     

Relations between hydrophobicity tested by three methods and surface chemical composition of Escherichia coli

The cell surface hydrophobicity of three strains of Escherichia coli cultured in liquid medium and on solid medium was measured using various methods including adsorption to pxylene, partition of cells in a polyethylene glycol/dextran (PEG/DEX) two phase system and contact angle measurements. The percentage adsorbed to pxylene ranged from 1.6% to 67% and the percentage of cells in polyethylene glycol phase ranged from 19% to 64%. The contact angle data of less than 40 degrees C revealed a hydrophylic character of the E. coli strains studied here. No relations were found between paraxylene/water partitioning, PEG/DEX partioning and water contact angles. The linear correlation coefficients between the results of the three hydrophobicity assays and the elemental concentration ratios obtained by X-ray photoelectron spectroscopy (XPS) were calculated. A linear correlation was found between the contact angles and the O/C ratios (r=0.91) and the N/C ratios (0.67). The adsorption to pxylene correlates better with N/C ratios (0.88) but does not correlate with O/C ratios (0.46). However, this test correlates with N/P ratios (0.79). No relation was obtained between partition in PEG/DEX system and any elemental concentration ratios. The surface composition determined by XPS was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbon-like compounds. The proteins/polysaccharides and the hydrocarbons/polysaccharides seems to determine the contact angle of E. coli but not the adsorption to paraxylene or partition in the PEG/DEX system.     

The effects of three non-antibiotic, antimicrobial agents on the surface hydrophobicity of certain micro-organisms evaluated by different methods

D.S. JONES, S.P. GORMAN, D.F. MCCAFFERTY AND A.D. WOOLFSON. 1991. The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.     

Bacterial hydrophobicity, an overall parameter for the measurement of adhesion potential to soil particles

The adhesion of Salmonella typhimurium to the mineral particles quartz, albite, feldspar, and magnetite was shown to correlate with the hydrophobicity of the cell surface as measured by hydrophobic interaction chromatography. The same effects were also seen for seven other selected test strains, including Streptococcus faecalis, Streptococcus faecium, Escherichia coli, Citrobacter freundii, Shigella sonnei, and Shigella boydii. When the test strain of Salmonella typhimurium, was repeatedly cultivated in Luria broth, thus selecting for different degrees of fimbriation and roughness of the cell surface, varied cell hydrophobicity but constant negative and positive charge values were obtained. High hydrophobicity values always coincided with enhanced adhesion to the mineral particles. The negative charge of the bacterial surface as measured by electrostatic interaction chromatography appeared to play no role in the adhesion event. However, the positive charges on the cell surface contributed to the adhesion process. This was especially evident for cells exhibiting a high degree of hydrophobicity. Alteration of the pH between 4 and 9 did not significantly affect the adhesion process.     

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.     

High surface hydrophobicity of hemagglutinatingVibrio cholerae and other vibrios

Surface hydrophobicity of hemagglutinatingVibrio cholerae, Vibrio parahaemolyticus, and NAG vibrios has been investigated. Most strains caused mannose-sensitive hemagglutination of monkey, guinea pig, chicken, and mannose-resistant hemagglutination of human erythrocytes with different degrees of hemagglutinating activity. Hemagglutinating strains adsorbed to a hydrophobic gel (Octyl Sepharose), whereas nonhemagglutinating strains failed to adsorb.Vibrio cholerae and other vibrios investigated seem to have pronounced surface hydrophobicity as estimated by Octyl Sepharose and they correspondingly autoaggregated into visible cell clumps in ammonium sulfate solution at low molarity (0.2–0.4 M). Nonhemagglutinating strains did not aggregate even at high (2 M) ammonium sulfate concentration. The presence of surface hemagglutinins of vibrios is growth-media-dependent. Strains, grown in four different liquid media, produced hemagglutinins and expressed pronounced surface hydrophobicity. Studies with electron microscopy revealed the presence of fimbriae on the vibrio cells. The number of fimbriae on the cells varied from strain to strain. Some strains possessed more than 300 fimbriae/cell whereas others had less than 10 fimbriae/cell. Vibrio hemagglutinins are easily detached from the cell surface by heating or sonication, and their cell surface hydrophobicity decreased simultaneously.     

Bacterial hydrophobicity, an overall parameter for the measurement of adhesion potential to soil particles.

The adhesion of Salmonella typhimurium to the mineral particles quartz, albite, feldspar, and magnetite was shown to correlate with the hydrophobicity of the cell surface as measured by hydrophobic interaction chromatography. The same effects were also seen for seven other selected test strains, including Streptococcus faecalis, Streptococcus faecium, Escherichia coli, Citrobacter freundii, Shigella sonnei, and Shigella boydii. When the test strain of Salmonella typhimurium, was repeatedly cultivated in Luria broth, thus selecting for different degrees of fimbriation and roughness of the cell surface, varied cell hydrophobicity but constant negative and positive charge values were obtained. High hydrophobicity values always coincided with enhanced adhesion to the mineral particles. The negative charge of the bacterial surface as measured by electrostatic interaction chromatography appeared to play no role in the adhesion event. However, the positive charges on the cell surface contributed to the adhesion process. This was especially evident for cells exhibiting a high degree of hydrophobicity. Alteration of the pH between 4 and 9 did not significantly affect the adhesion process.     

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.     

Change in the surface hydrophobicity of substrate cells during bdelloplast formation by Bdellovibrio bacteriovorus 109J.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J, the substrate cell surface becomes more hydrophobic. This was shown (i) by comparing the sensitivity to hydrophobic antibiotics of wild-type and lipopolysaccharide mutant strains of Salmonella typhimurium to that of the bdellovibrio growing on these strains and (ii) by measuring the binding efficiency of these strains, Escherichia coli, and their derived bdelloplasts to octyl Sepharose. The kinetics of increase in surface hydrophobicity was similar to the kinetics of the conversion of the substrate cell peptidoglycan to a lysozyme-resistant form (M. Thomashow and S. Rittenberg, J. Bacteriol. 135:1008-1014, 1978), and hydrophobicity reached a maximum at about 60 min in a synchronous culture. The change in hydrophobicity was inhibited by chloramphenicol, suggesting that bdellovibrio protein synthesis was required. Control experiments revealed that the free-swimming bdellovibrio had a more hydrophobic surface than the deep rough mutants of S. typhimurium.