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.     

Physicochemical and structural studies onAcinetobacter calcoaceticus RAG-1 and MR-481—Two standard strains in hydrophobicity tests

Acinetobacter calcoaceticus RAG-1 and MR-481, two standard strains used in microbial adhesion to hydrocarbons (MATH), were characterized by contact angles, pH-dependent zeta potentials, elemental surface composition by X-ray photoelectron spectroscopy (XPS), and molecular composition by infrared spectroscopy (IR). Negatively stained (methylamine tungstate) and ruthenium red-stained cells were studied by transmission electron microscopy to reveal the absence or presence of surface appendages. Despite the fact thatA. calcoaceticus RAG-1 is known to be extremely hydrophobic in MATH, whereas MR-481 is a completely non-hydrophobic mutant, neither XPS nor IR indicated a significant difference in chemical composition of the cell surfaces. Contact angles with polar liquids, water and formamide, were considerably higher on RAG-1 than on MR-481, in accordance with their relative hydrophobicities as measured by MATH. However, no significant differences in contact angles were observed between the two strains with apolar liquids like diiodomethane,-bromonaphthalene, and hexadecane. Fibrous extensions on RAG-1, observed after ruthenium red staining, were absent on the non-hydrophobic mutant MR-481. Tentatively, these extensions could be held responsible for the hydrophobicity ofA. calcoaceticus RAG-1.     

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.     

Unmasking of surface components by removal of cell-associated emulsan from Acinetobacter Sp. RAG-1

Summary Acinetobacter calcoaceticusRAG-1 cells lacking the emulsan capsule on the cell surface were obtained by two methods; a) by selecting for mutants that lack emulsan with a specific phage and b) by removal of the emulsan capsule from wild type cells with a specific emulsan depolymerase. Emulsan deficient cells obtained by either method become deficient in the adsorption of phage ap3 and sensitive to a newly isolated bacteriophage, nø. When RAG-1 cells were first treated with emulsan depolymerase and subsequently incubated without the enzyme, regeneration of the cell-associated emulsan was correlated with an increase in phage ap3 adsorption and an inhibition in phage nø adsorption. By partial regeneration of cell surface emulsan, a physiological state was obtained in which RAG-1 cells were sensitive to and efficiently adsorbed found phages. Enzyme-treated RAG-1 cells were found to be more adherent to hexadecane than the untreated RAG-1 cells. The data indicate that in addition to its function as the ap3 receptor, cell-associated emulsan masks the expression of other cell-surface determinant(s) which function(s) as: (i) receptor for bacteriophage nø, and (ii) cell-surface sites which enhance adherence to hydrophobic surfaces.Present address: Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA     

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.     

Alternate hydrophobic sites on the cell surface of Acinetobacter calcoaceticus RAG-1

Abstract Mutants of A. calcoaceticus RAG-1 lacking thin fimbriae (35 Å) do not adhere to hydrophobic surfaces [5], or grow on hydrocarbons under conditions of weak agitation and small inocula. Emulsan-deficient derivatives of such mutants, isolated in the present study, (i) lacked cell-surface emulsan, (ii) adhered avidly to hydrocarbons, (iii) lacked thin fimbriae, and (iv) regained the capacity to grow on hydrocarbons. The results show that emulsan masks an alternate hydrophobic site(s) on the cell surface of RAG-1.     

Adherence to HEp-2 cells and replication in macrophages of Salmonella derby of human origin.

Adherence to a HEp-2 cell monolayer was tested for in four strains of Salmonella derby which were isolated from patients with diarrhea. One strain, SB1, was highly adherent and another strain, SB4, was nonadherent. The other two strains exhibited moderate adherence. Further in vitro study of invasion of HEp-2 cells by S. derby and its replication in murine peritoneal macrophages was carried out using SB1 and SB4. Thin section electron micrographs revealed that SB1 invaded HEp-2 cells but SB4 did not. The number of viable bacteria within macrophages was determined at intervals after inoculation of bacteria. The result indicates that SB1 can replicate in the macrophages but SB4 cannot. Flagella and fimbriae were compared by electron microscopy between SB1 and SB4, and their lipopolysaccharides and outer membrane proteins were also compared with each other by SDS-polyacrylamide gel electrophoresis. The presence of a 41 kDa protein in the outer membranes of SB1 was only the difference detected, suggesting that this protein could be a factor required for adherence of this serovar to epithelial cells.     

Bacterial degradation of emulsan

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Bacterial degradation of emulsan.

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

New aspects of the role of MR/P fimbriae in Proteus mirabilis urinary tract infection

Proteus mirabilis, a common cause of urinary tract infection (UTI), produces a number of different fimbriae including mannose-resistant Proteus-like fimbriae (MR/P). The precise role of different P. mirabilis fimbriae in ascending UTI has not yet been elucidated. In this study, a clinical isolate of P. mirabilis and an isogenic mutant unable to express MR/P were tested using different experimental approaches. They were tested for their ability to cause infection in an ascending co-infection model of UTI and in a haematogenous model in the mouse. In both models, the mutant was less able than the wild-type strain to colonise the lower and upper urinary tracts although infectivity was not abolished. In vitro adherence to uroepithelial cells was also assessed. Significant differences in adherence between both strains were observed at 1 h but not at 15 min post infection. We have also shown that a wild-type strain carries two copies of the mrpA gene. These data reinforce the importance of MR/P fimbriae in P. mirabilis UTI although other virulence factors may be necessary for efficient colonisation and development of infection.     

Electron microscopy of adhesive interactions between Gardnerella vaginalis and vaginal epithelial cells, McCoy cells and human red blood cells

Exfoliated vaginal epithelial cells with attached bacteria, termed 'clue cells', which were procured from a patient with non-specific vaginitis, were stained with ruthenium red and examined by transmission electron microscopy. The attached bacteria appeared to adhere by means of an outer fibrillar coat. An epithelial tissue culture cell line (McCoy) and human red blood cells to which strains of Gardnerella vaginalis attached were similarly examined. The adherence of G. vaginalis to the epithelial cell line appeared to be mediated by an outer fibrillar coat while adherence to red cells appeared to be mediated by fimbriae. Transmission electron microscopy was performed on the Gardnerella strains used. Thin sections of tissue-culture-adherent strains revealed a dense outer fibrillar coat whereas the surface of the haemagglutinating strains showed fine fimbriae. Negative staining of haemagglutinating strains demonstrated fimbriae on a minority of organisms.     

Characterization of an outer membrane protein associated with haemagglutination and adhesive properties of enteroaggregative Escherichia coli O111:H12

Diarrhoeagenic Escherichia coli strains of serotype O111:H12 are characterized by their aggregative pattern of adherence on cultured epithelial cells and thus are considered enteroaggregative E. coli (EAEC). We have previously shown that these EAEC strains lack the genes encoding the aggregative fimbriae I and II described in other heterologous EAEC strains. In this paper, we show compelling data suggesting that a plasmid-encoded outer membrane 58 kDa protein termed aggregative protein 58 (Ap58) produced by EAEC O111:H12 strains, is associated with the adherence capabilities and haemagglutination of animal red blood cells. This conclusion is supported by several lines of evidence: (i) adherent O111:H12 strains are able to produce Ap58; (ii) non-adherent O111:H12 strains are unable to produce Ap58; (iii) antibodies raised against Ap58 inhibited adherence and haemagglutination of epithelial and bovine red blood cells, respectively; (iv) a non-adherent E. coli K-12 host strain containing the ap58 gene determinant on plasmid pVM15 displayed abundant adherence to cultured HEp-2 cells; and (v) the purified Ap58 bound specifically to HEp-2 and bovine red blood cells. Our findings indicate that the aggregative adherence in the O111:H12 strains may be also mediated by non-fimbrial adhesins. We believe our data contribute to the understanding of the adherence mechanisms of these organisms.     

Precise and rapid assessment of Escherichia coli adherence to vaginal epithelial cells by flow cytometry

BACKGROUND: In the pathogenesis of Escherichia coli urinary tract infections (UTIs) in women, infecting bacteria adhere to vaginal and periurethral epithelial cells prior to ascending to the bladder and causing infection. Complex interactions among specific bacterial adhesins and various host factors appear to influence adherence of E. coli to mucosal surfaces such as the urogenital epithelium. To conduct population-based studies assessing host epithelial cell determinants that influence bacterial attachment, a method of measuring bacterial adherence utilizing clinically derived epithelial cell samples is needed. METHODS: We developed and standardized an efficient, accurate, high-throughput method for analyzing the adherence of uropathogenic E. coli to clinical samples containing a large number of exfoliated vaginal epithelial cells (VEC). Three wild-type E. coli strains isolated from women with UTI (IA2 expressing pap-encoded, class II fimbriae only; F24 expressing pap-encoded, class II and type 1 fimbriae; and F20, without pap-encoded or type I fimbriae) were transformed with gfpmut3, encoding green fluorescent protein, incubated with VECs, and analyzed by flow cytometry. RESULTS: Enumeration of the binding of each E. coli strain to 10,000 VECs showed reproducible, highly significant strain-dependent differences in adherence to VECs. Differential analysis of the relative contributions of type 1 pili and P fimbrial-mediated binding to the adherence phenotype was performed. It demonstrated that IA2 binding was dependent entirely on P fimbriae, whereas F24 binding was dependent on both P and type 1 fimbriae. CONCLUSIONS: This method has great potential for use in high-throughput analyses of clinically derived epithelial cell samples and will be valuable in population-based investigations of host-parasite interactions in UTI utilizing VECs collected from specific patient groups.     

Surface structures, haemagglutination and cell surface hydrophobicity of Bacteroides fragilis strains.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2.1 +/- 0.25 nm and appeared to be 'curly'. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 Showed phase variation of fimbriae as expression of fimbriae was repressed at 20 degrees C and in early exponential phase at 37 degrees C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7.9 and 23.9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6.6 to 52.1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.     

Adhesive properties and antibiotic resistance of Klebsiella strains isolated from gastrointestinal tracts of children hospitalised in Wroclaw nad Opole

Gram negative Klebsiella bacilli present many pathogenic properties, which determine their ability to survive and rapid spreading in hospital environment. There are many factors responsible for the pathogenicity of Klebsiella strains: capsule, fimbriae, nonfimbrial adhesins, lipopolysaccharide of the cell wall and extracellular secreted exotoxins. Klebsiella strains are etiological agents of different nosocomial infections but also colonized gastrointestinal and respiratory tracts. The aim of our work were adhesive properties and antibiotic resistance of Klebsiella strains isolated from stool of hospitalized children, according to source of potential nosocomial infections--100 Klebsiella strains from Wroclaw and 76 strains from Opole, isolated in cases of diarrhea. The resistance of this strains to different group of antibiotics, the expression of ESBL enzymes, the activity in hemagglutination and their ability to adherence to different cell lines were tested. The highest resistance of all strains to aminopenicillins was observed. The production of ESBL was highest in strains from Opole (51% strains) then in Wroclaw (9%). In both hospital units, ESBL+ strains were resistant to aminoglicosides and cotrimoxazol but sensitive to ciprofloxacine. Using hemagglutination method the types of fimbriae were defined. Above 90% investigated Klebsiella strains showed the presence of fimbriae (in Wroc?aw more strains simultaneously expressed fimbriae type 1 and 3, in Opole mainly fimbriae type 3). Over 70% strains demonstrated the high level of adherence to cell lines. Only several strains showed the low level or the lack of adhesion. These results suggested that among Klebsiella strains in gastrointestinal tract were presented multiresistant strains with high ability to adherence, which may be potential source of nosocomial infections.     

Surface hydrophobicity and adherence of Candida to acrylic surfaces

The relationship between cell surface hydrophobicities and adherence capacities to acrylic surfaces was investigated with seven laboratory strains and eighteen clinical isolates of Candida species. C. albicans was less adherent to acrylic surfaces than were other species and hardly adhered to hexadecane, whereas other strains, which had a high affinity to hexadecane, were more adherent to acrylic surfaces. A correlation was observed between the adherence capacities of Candida species to acrylic surfaces and their cell hydrophobicities. When acrylic plates were coated with human whole saliva, the contact angle of the plate became smaller than that of the nontreated plate and adherence of hydrophobic strains decreased, whereas the adherence of C. albicans was not affected.     

Lack of correlation between fibrils, hydrophobicity and adhesion for strains of Streptococcus sanguis biotypes I and II

Fifteen strains of Streptococcus sanguis biotype I and eight strains of Streptococcus sanguis biotype II with peritrichous fibrils, tufts of fibrils or a mixture of fibrils and fimbriae on the cell surface, were tested for their ability to adhere to saliva coated spheroidal hydroxyapatite (S-SHA) in a radiolabelled assay. S. sanguis I strains adhered better than S. sanguis II strains and peritrichously fibrillar strains generally adhered better than tufted strains. There was no correlation between the density of fibrillation and adhesion. The only highly adherent strain of S. sanguis II carried fimbriae in addition to fibrils. No correlation was observed between cell surface hydrophobicity as measured by phase partitioning with hexadecane and adhesion to S-SHA.     

Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light.

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.     

Type I fimbriae mediate in vitro adherence of porcine F18ac+ enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (ETEC)

Type I fimbriae commonly expressed by Escherichia coli mediate initial attachment of bacteria to host epithelial cells. However, the role of type I fimbriae in the adherence of porcine enterotoxigenic E. coli (ETEC) to host receptors is unclear. In this study, we examined the role of type I fimbriae in the adherence and biofilm formation of F18ac+ ETEC by constructing mutant strains with deletion of type I fimbrial major subunit (fimA) or minor subunit (fimH). The data indicated that the isogenic ΔfimA and ΔfimH mutants showed significantly lower adherence to porcine epithelial IPEC-1 and IPEC-J2 cells as compared to the F18ac+ ETEC parent strain. In addition, the adherence of F18ac+ ETEC to both cell lines was blocked by the presence of 0.5% D-mannose in the cell culture medium. In addition, both mutant strains impaired their ability to form biofilm in vitro. Interestingly, the deletion of fimA or fimH genes resulted in remarkable up-regulation of the expression of adhesin involved in diffuse adherence (AIDA-I). These results indicated that type I fimbriae may be required for efficient adherence of F18ac+ ETEC to pig epithelial cells and, perhaps, biofilm formation.     

Occurrence of the Fimbria Gene hifA in clinical isolates of nonencapsulated Haemophilus influenzae

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA-positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.