Isolation and study of fosfomycin-resistant mutants of group A and B streptococci

Eighty-nine fosfomycin-resistant mutants of group A and B streptococci were isolated. The mutants differed essentially from the parent strains in cell-wall characteristics, such as morphology of cocci and chains, sensitivity to detergents and hydrophobic properties. At the same time, the recipient activity of the investigated mutants was not changed. It is supposed that resistance of mutants to fosfomycin is connected with a transport block of glycerol 3-phosphate--important lipoteichoic acid (LTA) structural component. Fosr mutants can be useful for experimental examination of Beachey's hypothesis about the importance of LTA or its complex with the M protein for adhesion.     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

Adherence of group B streptococci to human buccal epithelial cells,its dependence on the biological state of the culture

The adherence activity of Streptococcus agalactiae (group B) strains is directly dependent on the biological state of cultures. The aim of the present paper was to consider the effect of repeated transfers of cultures alternately on solid and liquid media and the effect of the growth phase. The strains, adhering weakly, strongly and variably to epithelial cells were employed in studies on the effect of repeated transfer. The percentage of adherent epithelial cells differed significantly after the first or the second transfer. On storage of the strains after the 3rd transfer at 4 degrees C for 4 d, the adherence activity decreased to the level of non-transferred strains. Ultrastructural analysis revealed in all strains the presence of capsular material, its character being similar both in strongly and in weakly adhering cultures. In weakly adherent strains, the structure of the capsular layer has not changed during transfer whereas the adherence of the strain increased considerably. The effect of the growth phase was studied during 3-48 h of incubation. The growth phase did not influence the adherence activity in strains that had been allowed to multiply for 3-24 h. After a long-term multiplication beyond 24 h, the adherence activity gradually decreased.     

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.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower ( P < 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower (P less than 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.     

Cell-surface hydrophobicity of adherent oral bacteria

Adherent bacteria were released from the surfaces of four freshly extracted teeth by mild sonic oscillation, and screened for cell-surface hydrophobicity on the basis of their ability to adhere to hexadecane. Of the 103 tooth isolates examined, 82 adhered to the test hydrocarbon. Hydrophobic bacteria could similarly be isolated from the stainless steel dental matrix bands following brief incubation in the mouth of a volunteer; 30 of 52 isolates examined adhered to hexadecane. Among those strains which adhered to hexadecane, streptococci were the most frequent type isolated. Various other morphological types were also observed, including cocci, bacilli, coryneforms, and filamentous bacteria. The high overall proportion of hydrophobic bacteria found in this study (72%) suggests that cell-surface hydrophobicity may play a role in adherence of certain oral species to the tooth surface.     

Inhibition of adhesive activity of K88 fibrillae by peptides derived from the K88 adhesin.

A cyanogen bromide fragment derived from the K88ab adhesin inhibited the hemagglutinating activity of K88 fibrillae. Smaller fragments which inhibited the adherence of K88 fibrillae to erythrocytes or to intestinal epithelial cells were obtained by digestion of K88ab fibrillae with alpha-chymotrypsin. Active peptides were isolated from the digestion mixture and identified as Ser-Leu-Phe and Ala-Ile-Phe. Both tripeptides correspond to the peptide stretches Ser-148-Leu-Phe-150 and Ala-156-Ile-Phe-158, respectively, which are part of conserved regions in the primary structure of the K88 variants ab, ac, and ad. The isolated tripeptides inhibited the hemagglutinating activity of purified K88 fibrillae in the 1 to 5 microM range, while adherence of the fibrillae to intestinal epithelial cell brush borders was inhibited in the 10 to 50 microM range. Furthermore, the tripeptides were capable of eluting attached bacteria from agglutinated erythrocytes. The inhibitory activity of the isolated peptides was confirmed by testing various synthetic peptides for their ability to inhibit the interaction of the different K88 variants with various species of erythrocytes. The significance of these findings for the localization of the receptor-binding domain is discussed.     

The role of surface structures of Streptococcus agalactiae in adhesion to epithelial cells

Induced mutants of S. agalactiae which differed in surface structures were used for the study. The aim of using them was to try to correlate the presence of defined structures or surface properties with the ability of group B streptococci to attach to epithelial cells. The presence of protein antigen R conditioned strong binding of S. agalactiae cells to hydrophobic gel. Strains bearing clumping factor (CF) showed high surface hydrophobicity and presented compact growth in serum soft agar. However, there was no correlation between high surface hydrophobicity and the ability to adhere. Fibrinogen binding decreased the attachment to epithelial cells of CF-positive strains. Preincubation of bacterial cells with lectin (ConA) did not influence the attachment of S. agalactiae strains with protein surface antigen but increased the adhesion of the strains with polysaccharide antigen or untypable.     

Surface free energy and interaction of Staphylococcus epidermidis with biomaterials

The adhesion of twenty nine Staphylococcus epidermidis strains to teflon, polyethylene, polycarbonate and bovine pericardium was studied in vitro and examined in relation to the surface free energies of both bacteria and biomaterials. All S. epidermidis strains had similar surface free energies, close to that of water, and adhered better to the materials with analogous surface free energies. There was a significant correlation (Kendall's Tau B = 1000) of biomaterial's surface free energy with the number of adhering bacteria. This correlation is inverse (Kendall's Tau B = -1000) when surface hydrophobicity is considered instead of surface free energy. This indicates that in Staphylococcus epidermidis adherence to biomaterials is inversely correlated to the surface hydrophobicity of the last, being so just the opposite of that occurring with other bacteria.     

Bacterial adherence to polystyrene: a replica method of screening for bacterial hydrophobicity.

A simple replica method is described for the rapid identification of colonies of bacteria which adhere to polystyrene. A correlation was found between the adherence of bacterial strains to polystyrene and cell surface hydrophobicity, suggesting the use of this technique in screening for cell surface mutants and in the isolation of hydrophobic bacteria from nature.     

Multiple changes in cell wall antigens of isogenic mutants of Streptococcus mutans.

Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.     

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.     

Influence of aeration of Candida albicans during culturing on their surface aggregation in the presence of adhering Streptococcus gordonii

Candida albicans surfaces are extremely sensitive to changes in growth conditions. In this study, adhesion to glass of aerated and non-aerated C. albicans ATCC 10261 in the presence and absence of adhering Streptococcus gordonii NCTC 7869 was determined in a parallel plate flow chamber. In addition, the influence of aeration on the yeast cell surface hydrophobicity, surface charge, and elemental cell surface composition was measured. S. gordonii adhering at the glass surface caused a reduction in the initial deposition rate of C. albicans, regardless of aeration. In a stationary end-point, only adhesion of non-aerated C. albicans was suppressed by the adhering S. gordonii. Non-aerated yeasts had a higher O/C elemental surface concentration ratio, indicative of cell surface polysaccharides, than aerated yeasts, at the expense of nitrogen-rich cell surface proteins. Both yeasts were essentially uncharged, but the nitrogen-rich cell surface of aerated yeasts had a slightly higher water contact angle than non-aerated yeasts. Summarizing, this study suggests that highly localized, hydrophobic cell surface proteins on C. albicans are a prerequisite for their interaction with adhering streptococci.     

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.     

Microplate fluorescence assay for measurement of the ability of strains of Listeria monocytogenes from meat and meat-processing plants to adhere to abiotic surfaces

Listeria monocytogenes is a significant food-borne pathogen that is capable of adhering to and producing biofilms on processing equipment, making it difficult to eliminate from meat-processing environments and allowing potential contamination of ready-to-eat (RTE) products. We devised a fluorescence-based microplate method for screening isolates of L. monocytogenes for the ability to adhere to abiotic surfaces. Strains of L. monocytogenes were incubated for 2 days at 30 degrees C in 96-well microplates, and the plates were washed in a plate washer. The retained cells were incubated for 15 min at 25 degrees C with 5,6-carboxyfluorescein diacetate and washed again, and then the fluorescence was read with a plate reader. Several enzymatic treatments (protease, lipase, and cellulase) were effective in releasing adherent cells from the microplates, and this process was used for quantitation on microbiological media. Strongly adherent strains of L. monocytogenes were identified that had 15,000-fold-higher levels of fluorescence and 100,000-fold-higher plate counts in attachment assays than weakly adherent strains. Strongly adherent strains of L. monocytogenes adhered equally well to four different substrates (glass, plastic, rubber, and stainless steel); showed high-level attachment on microplates at 10, 20, 30, and 40 degrees C; and showed significant differences from weakly adherent strains when examined by scanning electron microscopy. A greater incidence of strong adherence was observed for strains isolated from RTE meats than for those isolated from environmental surfaces. Analysis of surface adherence among Listeria isolates from processing environments may provide a better understanding of the molecular mechanisms involved in attachment and suggest solutions to eliminate them from food-processing environments.     

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.     

The correlation between surface hydrophobicity and adherence of Bifidobacterium strains from centenarians' faeces

To develop food-grade bifidobacteria micro-ecologics, screening for Bifidobacteria strains which can adhere to intestinal epithelial cells was finished. Twenty-three bifidobacterial strains tested were isolated from centenarians in Bama country, the fifth long-lived district in the world. Surface hydrophobicity and adherence capability to intestinal epithelial cells in vitro of bifidobacteria were simultaneously investigated for the first time. It has been demonstrated that all the strains exhibited adhesive properties to some extent using intestinal Caco-2 cell line in in vitro model. It could be conclude that the higher hydrophobic strains the stronger adhesive capability. The highest value of hydrophobicity (37.24+/-1.45% and 32.06+/-1.21%) was obtained for strains H-10 and I-6, respectively; correspondingly, the strongest adherence ability (49.47+/-4.88/cell and 47.33+/-2.72/cell) was achieved, respectively. Correlation between surface hydrophobicity and adherence ability of different Bifidobacterium strains including polynomial regression equation (R2=0.78) had been achieved. The present study provided a liable and effective method for screening bifidobacteria with the ability to adhere to intestinal epithelial cells.     

Effect of aminoglycosides on surface hydrophobicity of Acinetobacter baumannii

Effects of amikacin, gentamicin, netilmicin and tobramycin at subinhibitory concentrations (sub-MICs) (11/4, 1/8, 1/16 or 1/32 of their MICs) on the cell surface hydrophobicity of two Acinetobacter baumannii strains (7194 and 16265) were evaluated. Hydrophobicity was determined by two different methods - by adherence of bacteria to hydrocarbon (xylene) and by aggregation of bacteria in ammonium sulphate solutions at various concentrations. The adherence of A. baumannii strains to xylene decreased, mainly, after treatment with netilmicin at 1/4, 1/8 or 1/16 of the MIC (to 6.4%, 17.0% or 24.5% of the control value) (strain 7194) and after treatment with amikacin and gentamicin at 1/4 of their MICs (to 58.4% or 54.4%) (strain 16265). A decrease in surface hydrophobicity of exposed strains under these conditions was shown in salting-out test, too. Tobramycin reduced hydrophobic properties of A. baumannii strains at all tested sub-MICs to only a small extent.