CURRENT MICROBIOLOGY Vol. 25 (1992), pp. 9-17

Current Microbiology 9 Springer-Verlag New York Inc. 1992

Demonstration of Exopolysaccharide Production by Enterohemorrhagic Escherichia coli Alan D. Junkins 1 and Michael P. Doyle 2 Food Research Institute and Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA

Abstract. Enterohemorrhagic Escherichia coli O157:H7 produces visibly slimy colonies when grown on Sorbitol/MacConkey or Maloney's agar plates at room temperature, indicative of exopolysaccharide (EPS) production. Eighteen of 27 (67%) wild-type E. coli O157:H7 isolates produced enough EPS to be visually distinguishable. Of five strains that showed no visible EPS production on these media, four (80%) did produce slimy colonies on media containing higher salt concentrations. Measurements of EPS production by colorimetric determination of uronic acid indicated that EPS production was affected by growth temperature, atmosphere, and medium. Wild-type E. coli O157:H7 strain 932 produced the greatest amounts of EPS when grown anaerobically at 37~ whereas its plasmid-cured derivative 932P produced large quantities of EPS when grown aerobically at room temperature. Electron micrographs revealed thin, flexible fibers extending from the bacterial cell surface. Cells of strain 932P grown aerobically at room temperature were completely encased in a thick EPS matrix. Chemical analysis of purified EPS revealed that it is very similar or identical to colanic acid. E. coli O157:H7 adheres better to INT 407 cells when grown under conditions that favor high EPS production than when grown under conditions that repress EPS production.

The role of bacterial exopolysaccharides (EPS) in the pathogenesis of disease has been well documented. The ability of EPS-producing strains to evade phagocytic mechanisms and fixation by serum complement have made them an almost universal virulence factor among bacteria that cause invasive infections [5, 18]. Bacterial EPS have been implicated as an adherence factor both to inert and cellular surfaces [3, 9, 15, 17, 19]. Some investigators have suggested that almost all bacteria produce EPS in their natural environments [4]. However, production is typically lost quickly when cultured on artificial bacteriological media. For this reason, many bacteria have been identified as non-EPS-producing, although it is likely that these organisms are capable of producing EPS in vivo. Enterohemorrhagic Escherichia coli (EHEC), i Present address: Department of Medical Laboratory Sciences, Medical University of South Carolina, Charleston, SC 29425, USA. 2 Present address: Food Safety and Quality Enhancement Laboratory, University of Georgia, Georgia Station, Griffin, GA 30223, USA.

particularly those of the serotype O157:H7, have been associated with hemorrhagic colitis and hemolytic uremic syndrome since 1982. Several studies have addressed the surface characteristics of these organisms [22], but surface polysaccharides have not been identified. Studies on adherence of the organism to intestinal epithelium have failed to conclusively identify any adhesin. A plasmid-encoded fimbria has been recognized [11], but the importance of this fimbria in mediating adherence is questionable [21, 22, 26, 27]. The purpose of this study was to examine EHEC strains for production of EPS, to determine the effect of culture conditions on EPS production, and to purify and characterize the structure of EPS. Materials and Methods Bacterial strains. E. coli O157:H7 strains 932 and 932P were furnished by the Centers for Disease Control (Atlanta, Georgia). Strain 932P was derived from strain 932 by ethidium bromide treatment. Media. Maloney's agar [14] consisted of 10 g Na2HPO4, 0.9 g

KC1, 0.6 g MgSO 4 9 7H20, 10 g casamino acids, 5 g glucose, and

Address reprint requests to: Dr. Michael P. Doyle, Food Safety and Quality Enhancement Laboratory, University of Georgia, Georgia Station, Griffin, GA 30223, USA.

10 15 g agar/L. MacConkey/Sorbitol (SMAC) agar was prepared from a commercial sagarless MacConkey agar base (Difco Laboratories, Detroit, Michigan) supplemented with 1% sorbitol. Penassay and tryptic soy broths were prepared from commercial powders (Difco).

Visual estimation of EPS production. Bacteria were inoculated onto plates of different media and incubated at room temperature or 37~ for up to 12 days. EPS production was considered positive if one or more colonies were visibly slimy or mucoid.

Quantitative determination of relative amounts of EPS produced. Bacteria were grown on agar surfaces under different culture conditions. After 24-48 h of incubation, the lawn of growth was resuspended in 10 ml of 0.15 M NaCI. The amount of uronic acid in the suspension was determined by the colorimetric assay for uronic acid described by Blumenkrantz and Asboe-Hansen [2], with glucuronic acid as a standard. The BCA assay for protein (Pierce, Rockford, Illinois) was performed to account for differences in quantities of bacterial growth. A UA/P ratio, defined as the micrograms of uronic acid per milligram of protein, was determined for each suspension.

Electron microscopy. Bacteria were grown under a variety of conditions. Growth from an agar surface, or the pellet from a 10-ml penassay broth culture was resuspended in 2 ml of fixative consisting of 2.5% glutaraldehyde, 100 mM lysine, and 0.075% ruthenium red (RR), in 0.1 M cacodylate buffer, pH 7.0 (CB), and agitated gently for 20 rain at room temperature. Cells were sedimented by centrifugation at 1500 g for 10 min at room temperature. Pellets were resuspended in 2 ml of the same fixative solution without lysiue. Suspensions were gently agitated for 100 min at room temperature. Alternatively, bacterial growth was suspended in 1 ml undiluted rabbit serum raised against a crude EPS preparation and incubated at room temperature for 1 h. This suspension was centrifuged as above, and the pellet was resuspended in 2 ml fixative containing 0.5% glutaraldehyde, 0.15% RR, and 100 mM lysine in CB and incubated at room temperature for 30 min. All samples were treated the same after the fixation steps. Suspensions were centrifuged at 1500 g for 10 rain, and pellets were resuspended in a molten 4% agarose solution held at 40~ After the agarose had hardened, it was cut into blocks approximately 2 mm 3. These blocks were treated with 3% glutaraldehyde and 0.05% RR in CB at 4~ for 4 h. Agarose blocks were washed five times with 2 ml 0.05% RR in CB for 10 min per wash before treatment with 2% osmium tetroxide for 2 h at room temperature, and the blocks were again washed as before. A graded sequence of 30, 50, 70, and 90% acetone solutions was used to dehydrate the blocks, with 30 min of agitation at room temperature at each acetone concentration. Dehydration was completed by four 15-min exposures to 100% acetone. Infiltation of embedding resin was accomplished by the following sequence of treatments: (a) 3 : 1 acetone:unaccelerated Durcupan, 60~ 2 h; (b) 1 : 1 acetone: unaccelerated Durcupan, 60~ 2 h; (c) 1 : 3 acetone: unaccelerated Durcupan, 60~ 2 h; (d) two treatments with 100% unaccelerated Durcupan, 60~ 2 h each; (e) two treatments with 100% accelerated Durcupan, 60~ 1.5 h each. Agar blocks were cleaned of excess resin on blotting paper and transferred into Beem capsules, which were filled with 100% accelerated Durcupan. Resin was allowed to cure at 60~ for 4 days. Blocks were

CURRENT MICROBIOLOGY Vol. 25 (1992)

trimmed and cut into 8-/zm sections, which were stained with uranyl acetate and lead citrate and viewed in a Hitachi HT600 transmission electron microscope. EPS purification. Strain 932P was grown on Maloney's agar aerobically at room temperature for 48-72 h. Growth was resuspended in 1% phenol and transferred to a 250-ml centrifuge bottle. The suspension was shaken well for 10 min to break up all clumps. Cells were sedimented by centrifugation at 16,000 g for 5 h at 4~ The supernatant solution was mixed with 1.5 volumes of acetone prechilled to -70~ Spools of precipitate collected on the ends of Pasteur pipettes were dehydrated for 3-5 min in cold ( - 70~ acetone, then air dried for about 10 min. The precipitate was dissolved in 200 ml of 0.02% sodium azide in either water or 0.15 M NaC1 by stirring at room temperature for 8-24 h. A portion of this solution was labeled "crude EPS" and set aside for comparison purposes. Hexadecyltrimethyl ammonium bromide (HTAB, Sigma Chemical Company, St. Louis, Missouri) (4 g) was added to the remainder of the solution, which was gently stirred for about 5 min. This mixture was centrifuged at 16,000 g for 30 rain at 20~ and the pellet was resuspended in 200 ml of 1 M NaC1 by shaking at room temperature for 24-36 h. Acetone precipitation was repeated as before. Spools were dissolved in 32.5 ml of 4 M guanidine hydrochloride, 5 mM Na2EDTA , 10 mM sodium phosphate, pH 6.5. Cesium chloride density gradient ultracentrifugation was then carried out as described by Rose [20]. Crystalline CsCI2 was added to achieve a density of 1.42 g/ ml. The solution was then transferred into a 38.5-ml Ultra-Clear ultracentrifuge tube (Beckman Instruments, Palo Alto, California), and the solution was overlaid with light mineral oil to fill the tube. This preparation was centrifuged at 76,000 g at 20~ for 72-80 h in a type 60 Ti rotor in a Beckman L5-65 ultracentrifuge. Fractions, approximately 1 ml each, were collected from the bottom of the tube through a 22-gauge needle. Peaks were detected by UV absorbance at 270 nm. Fractions containing EPS were pooled and dialyzed extensively against 50 mM ammonium bicarbonate at 4~ and lyophilized.

Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was done according to the procedure of Laemmli [12] with a BioRad Mini PROTEAN II apparatus. Gets were stained with Coomassie brilliant blue, periodic acid-Schiff, the alcian blue method of Wardi and Michos [28], or the silver stain for lipopolysaccharide as decribed by Tsai and Frasch [25]. Chemical characterization of EPS. Samples to be analyzed for chemical composition were purified according to the procedures described above. They were redissolved in double-distilled, deionized water and lyophilized for a second time to ensure removal of ammonium bicarbonate. EPS structure was analyzed at the Complex Carbohydrate Research Center (University of Georgia) by gas chromatography, mass spectrophotometry, and nuclear magnetic resonance. Visual adherence assay. Strains 932 and 932P were grown anaerobically on Maloney's agar without glucose and Maloney's agar plus 1.5% glucose for 18-24 h at 37~ Growth from the plates was resuspended in 10 ml sterile TBS. Optical density at 640 nm of the suspension was adjusted to 0.5, equal to approximately 3.5 x 108 CFU/ml. This suspension (2 ml) was applied to monolayers of INT 407 cells grown on 18-mm cover slips in 35-mm petri

A.D. Junkins and M.P. Doyle: Exopolysaccharide Production by EHEC dishes. The suspensions were incubated over the monolayers for 1 h at 37~ without agitation. Monolayers were washed five times with sterile TBS containing0.1% Tween 20 and allowed to air dry for about 10 min. Cover slips were removed from the petri dishes and inverted on a glass slide containinga drop of an 8% dilution of commercial strength Giemsa stain (Fisher Scientific, Fair Lawn, New Jersey). Monolayers were viewed by phase contrast microscopy, and the number of bacteria adhering to 20-30 INT 407 cells was counted.

Results Demonstration of EPS production. Visible EPS production was initially observed on plates of SMAC agar that were inoculated with E. coli O157:H7 strains 932 and 932P and incubated at room temperature for several days. Moist colonies became visible after 5 days. Large quantities of EPS were evident when colonies of strain 932P were held longer at room temperature (Fig. la). More rapid EPS production occurred on Maloney's agar [14] than on SMAC agar, particularly by plasmid-cured strains, which produced visibly slimy colonies on this medium throughout the growth cycle (Fig. lb). Rates of EPS production varied among E H E C isolates (Table 1). EPS was produced within 5 days by 11 of 27 wild-type strains, and between 8 and 12 days for seven other strains. Both strains ofplasmidcured E. coli O157:H7 and five of six non-O157:H7 E H E C strains produced visible EPS on both media within 5 days (Fig. 1). Five E. coli O157:H7 wild-type strains that had no visible slime formation on these media within 5 days were streaked onto plates of Maloney's agar containing 0, 115 raM, or 265 m M of added NaCI. Taking into account the 85 mM total concentration of all salts in Maloney's agar, these media contained concentrations of total salts of 85,200, and 350 mM, respectively. Four of five and three of five strains produced visible EPS when grown at room temperature for 48 h and at 37~ for 24 h, respectively. Strains were more likely to produce visible EPS at the higher salt concentrations. Effect of growth temperature and atmosphere on EPS production. Uronic acids are c o m m o n constituents of bacterial exopolysaccharides and are not found in other bacterial structures. Therefore, uronic acid is a much more specific indicator of EPS production than other assays for carbohydrate. Production of EPS was monitored by quantitative determination of uronic acid per milligram of protein. When grown at room temperature, strain 932P pro-

11

duced significantly more EPS than did its parental strain (Table 2). At this temperature EPS production by both strains appeared to be enhanced under aerobic conditions, although differences were not statistically significant (p > 0.05). When grown at 37~ EPS production by strain 932 was equal to or greater than that of strain 932P. Production of EPS by strain at 37~ was enhanced under anaerobic atmosphere. UA/P measurements of strains 932 and 932P grown anaerobically in penassay broth at 37~ were not significantly greater than zero.

Visualization of EPS on bacterial cell surfaces. Electron microscopic examination of bacterial EPS is complicated by the high water-holding capacity [23] and polyanionic quality of the molecules. This typically causes the EPS structure to collapse during the dehydration and infiltration steps in preparation for electron microscopic observation [4]. This explains why EPS is usually not seen in electron micrographs unless steps are taken to prevent dehydration. The EPS structure can be stabilized by treatment of the samples with agents that can form cross-bridges between EPS fibers, such as lysine or anti-EPS serum [13]. Our studies indicate that EPS produced by E. coli O157:H7 is prone to collapse unless stabilized, preferably by EPS-specific serum. When stabilized, it is apparent that EPS is a major cell surface structure. Electron micrographs of lysine-stabilized 932 cells grown aerobically at room temperature for 48 h on Maloney's agar revealed ruthenium redstained fibrils extending from the cell surface (Fig. 2a). These very thin and flexible structures tended to conglomerate, often distant from the bacterial cell. The fibers appeared to link the cells to each other in microcolonies. Lysine proved incapable of preventing dehydration of the EPS structure when strain 932P was grown under conditions that encourage very high levels of EPS production. When serum raised against a crude EPS preparation was used to stabilize the EPS structure, electron micrographs revealed bacterial cells completely encased within ruthenium red-stained material (Fig. 2b). Individual fibers of EPS were also visible. Bacterial pellets from broth cultures were also processed for electron microscopy. Both strains 932 and 932P grown anaerobically at 37~ in penassay broth had EPS fibers on the cell surface (data not shown). Under these conditions, it appeared that strain 932 produced more EPS than did strain 932P. The observation of EPS in these samples indicates

12

CURRENT MICROBIOLOGY VO1. 25 (1992)

Fig. 1. E. coli O157:H7 strain 932P grown on (A) Sorbitol/MacConkey (SMAC) agar aerobically for 15 days at room temperature, and (B) Maloney's agar aerobically for 2 days at 25~

that even when UA/P ratios are very small, low level EPS production still occurs. Purification and characterization of EPS. Attempts to purify EPS from the surface of strain 932P were complicated by the strong tendency of the polysaccharide to nonspecifically bind proteins and lipopolysaccharide. High salt concentations were successful at breaking these nonspecific attractions, hence the guanidine hydrochloride/cesium chloride density gradient technique described by Rose [20] was utilized. UV absorbance analysis of fractions demonstated a large central peak (Fig. 3). When the uronic acid assay was done on the fractions, pink color was evident in fractions from the large central peak, indicating the presence of uronic acid-

containing EPS. When fractions were analyzed by SDS-PAGE, the large central peak was determined to contain pure EPS (Fig. 4). LPS typically was detected in lower density fractions. Purified EPS separated by SDS-PAGE (Fig. 4) did not react with the Coomassie brilliant blue stain even after gels were dehydrated by the method of Yan [29] to increase sensitivity. In addition, purified EPS did not react with the silver stain for lipopolysaccharide [25], indicating an absence of LPS in the pure sample. EPS was clearly stained by the alcian blue method of Wardi and Michos [28]. This stain produced a fairly distinct band with a molecular weight greater than 200,000, which smeared up from there, completely through the 4% polyacrylamide stacking gel to the bottom of the well. In highly

A.D. Junkins and M.P. Doyle: Exopolysaccharide Production by E H E C

13

Table 1. Visible EPS production when grown at room temperature for 5 or 12 days a N u m b e r (%)

positive Number

Group E. coli O157:H7 wild-type clinical isolates E. coli O157:H7 wild-type meat isolates E. coli O157:H7 plasmidcured isolates Non-O157 enterohemorrhagic E. coli

of strains

5 days

12 days

21

7 (33)

13 (62)

6

4 (67)

5 (83)

2

2 (100)

2 (100)

6

5 (83)

5 (83)

a Grown on SMAC and Maloney's agars.

Table 2. Effect of growth atmosphere and temperature on UA/P" Mean UA/P -+ SEM Strain 932

Atmosphere b Anaerobic Microaerobic Aerobic

932 P

Anaerobic Microaerobic Aerobic

RT/48h C 5.4 9.9 13.1 29.5 23.3 74.8

_+ -+ -+ + -+ _+

0.8 5.3 6.8 17.3 14.9 18.4

37~C/24h J 7.6 3.3 3.9 5.1 2.2 2.3

_+ 1.8 e -+ 0.9 _+ 1.8 _+ 2.6 +_ 0.5 + 1.3

a UA/P, micrograms uronic acid per milligram protein. Means of three determinations. b Anaerobic: 10% CO2, 10% Hi, 80% N2 ; microaerobic: 5% 02, 10% CO2, 85% N2; aerobic: normal atmosphere. c RT/48h, bacteria grown on Maloney's agar at room temperature for 48 h. d 37oc/24h, bacteria grown on Maloney's agar at 37~ for 24 h. e p < 0.05, significantly different from 932/aerobic.

Fig. 2, Electron micrograph o f E . coli O157:H7 strains grown on Maloney's agar aerobically for 3 days at room temperature. (A)

Strain 932, lysine-stabilized; (B) strain 932P, serum-stabilized. Bar, 0.6 txm.

concentrated samples, some smearing below the high molecular weight band also occurred. This suggests a polydisperse distribution of molecular weight. A similar, though much more weakly stained, pattern was obtained with the periodic acid/ Schiff stain for carbohydrate. Chemical structure of EPS. Compositional analysis of purified EPS was accomplished through gas chromatography and mass spectrometry. EPS consists of fucose, galactose, glucose, and glucuronic acid, which make up approximately 90% of the total weight (Table 3). For comparison, the calcu-

lated percentage composition of the colanic acid subunit [24] also is included in Table 3. Results of analysis of permethylated glycosyl residues of EPS by gas chromatography and mass spectrometry are shown in Table 4. These data indicate that all four sugars are present in unbranched form and that fucose and galactose also demonstrate some branching. Nuclear magnetic resonance analysis of EPS (data not shown) was hampered by the high viscosity of the solution, which caused broadening of peaks. However, peaks at 1.44 ppm and 2.18-2.21 ppm indicated the presence of acetyl and pyruvyl

14

CURRENT MICROBIOLOGY VoL 25 (1992)

3

Table 3. Glycosyl composition of EPS purified from E. coli strain 932P compared with calculated composition of colanic acid hexasaccharide subunit

-1.65 -1.6

2.5

"1.55 ~,

2

-1.5

-~1.5

1.45

>,

-1,4 -1.35 0.5

-1.3

o

.4

1'o 1'5 2b 2'5 3'0 3~ 4'o 45

Percent of total weight

E

1,25

Glycosyl residue

Strain 932P EPS a

Colanic aci&

Fucose Galactose Glucose Glucuronic acid

32.2 21.4 16.2 16.2

29.4 29.4 14.7 14.7

Total carbohydrate

88.2

88.2

Fraction Number

Fig. 3. Density and UV absorbance read at 270 nm of fractions from guanidine hydrochloride/cesium chloride density gradient ultracentrifugation of EPS from E. coli O157:H7 strain 932P. Line, density in g/ml; filled squares, absorbance at 270 nm.

Mean of two samples analyzed by gas chromatography and mass spectrometry. Neutral residues quantitated by alditol acetate procedure. Glucuronic acid quantitated by TMS methylglycoside procedure. b Values calculated from colanic acid subunit structure proposed by Sutherland [24].

Fig. 4. SDS-12% polyacrylamide gel of crude and purified EPS preparations. Lane 1, low-molecular-weight protein standards (molecular weights: 21,500, 31,000, 45,000, 66,200, and 97,400 Da); lane 2, high-molecular-weight protein standards (molecular weights: 45,000, 66,200, 97,400, 116,250, and 200,000 Da); lanes 3, 5, 7, and 9, crude EPS prepared by acetone precipitation of a phenol extract of E. coli O157:H7 strain 932P; lanes 4, 6, 8, and 10, purified EPS prepared as described in text. Lanes 1-4, stained with Coomassie brilliant blue; lanes 5 and 6, stained with alcian blue procedure of Wardi & Michos [28]; lanes 7 and 8, stained with periodic acid-Schiff reagent; lanes 9 and 10, stained with silver stain for LPS as described by Tsai and Frasch [25].

A.D. Junkins and M.P. Doyle: Exopolysaccharide Production by E H E C

Table 4. Glycosyl linkages of EPS purified from E. coli strain 932P a Glycosyl residue Fucose Glucose Galactose Glucuronic acid

Position of methyl groups

Glycosyl linkage

Percent of residue total b

2,3 2 2, 4, 6 2, 4, 6 2, 3 2, 3

4-1inked 3,4-1inked 3-1inked 3-1inked 4,6-1inked 4-1inked

6.4 8.5 27 22 20 17

a Determined by methylation of alditol acetates with methyl iodide followed by gas c h r o m a t o g r a p h y and m a s s spectrometry. I, Percent of total residue expected based on compositional analysis results.

Table 5. A d h e r e n c e of E. coli strains 932 and 932P grown anaerobically at 37~ on M a l o n e y ' s agar to I N T 407 cells c' Strain

Glucose b

932 932P

0% 1.5% 0% 1.5%

M e a n UA/P -+ SD ' 1.54 8.16 1.59 11.88

-+ 0.44 --~ 0.41 e -+ 0.20 + 1.17 e

Mean B/C -+ SD a 2.70 8.49 3.91 9.06

-+ 1.03 + 1.50 f +- 0.57 + 2.70 /

M e a n s of four determinations. b Glucose concentration in medium. c UA/P, micrograms uronic acid per milligram protein; SD, standard deviation. d B/C, bacteria per I N T 407 cell. e p < 0.01. f p < 0.05.

substituents. Therefore, it is likely that the EPS contained both acetyl and pyruvyl groups.

Adherence to INT 407 monolayers. To test the effect of EPS production on ability to adhere to INT 407 cells, bacteria were grown anaerobically at 37~ on two different variations of Maloney's agar. Previous results (unpublished data) had indicated that, when strains 932 and 932P were grown anaerobically, low levels of EPS would be produced on Maloney' s agar with no glucose, whereas higher levels would result when glucose was added at a final concentration of 1.5%. Although other culture conditions would be expected to yield even higher levels of EPS production, these media were chosen because they would allow for a large increase in EPS production with as little change as possible in the medium composition or culture conditions. UA/P measurements of strains 932 and 932P grown under these conditions confirmed that significantly higher EPS production occured on the high-glucose medium (Table 5). Ac-

15

cording to a visual adherence assay, it appeared that EPS was capable of mediating or enhancing adherence to INT 407 cells. Both strains 932 and 932P adhered signifcantly more after growth on the high-glucose medium (Table 5). When compared with adherence of bacteria grown on no-glucose medium, strain 932 adhered 3.1 times more and strain 932P adhered 2.3 times more on high-glucose medium.

Discussion When grown under culture conditions that favor expression of EPS, 67% (18 of 27) E. coli O157:H7 wild-type strains produced visibly slimy colonies. However, EPS production also occurred on media even when colonies were not visibly slimy. Under conditions in which no uronic acid was measurable, electron microscopy still revealed the presence of EPS fibers on the cell surface. Therefore, it appears that EHEC cultures are capable of constitutive expression of low levels of EPS. Examination of the effects of culture conditions on EPS expression indicated that the polysaccharide is best produced at room temperature aerobically, particularly by plasmid-cured strains. The wild-type strain 932 produced levels of EPS higher than the cured strain 932P when grown at 37~ anaerobically. Therefore, wild-type EHEC strains are capable of producing EPS under the temperature/atmosphere conditions that occur in vivo. Reasons for the differences in EPS expression by the parental and cured strains are not known. It is possible that the 60-MDa plasmid encodes some factor that either directly regulates EPS expression or in some indirect manner influences the ability of the cell to produce EPS. However, since the cured strain was derived by treatment with ethidium bromide, a known mutagen, it cannot be unequivocally stated that, disregarding the difference in plasmid content, the two strains are completely isogenic. A chromosomal mutation in either strain may be affecting EPS expression. Chemical analysis of purified EPS from strain 932P revealed the presence of fucose, galactose, glucose, and glucuronic acid (Table 3), along with acetyl and pyruvyl groups. These six components also compose the EPS known as colanic acid, which is produced by several strains of E. coli and Salmonella [7, 23, 24]. The glycosyl linkage data (Table 4) further indicate that the EPS from strain 932P is identical or very similar to the structure of colanic acid as proposed by Sutherland [24]:

16

CURREN~MICROBIOLOGYVol. 25 (1992) Pyruvate ~

Gal 1 4 GlcA 1

$/3 3 Gal 1 Acetyl 13 3/4 $ (---~ 3 Glc 1 ~ 4 F u c 1 ~ 4 F u c 1 ----~)

T h e r e f o r e , results of the c h e m i c a l analysis strongly suggest that the E P S f r o m strain 932P is colanic acid. V e r y little is k n o w n a b o u t the function o f colanic acid. It does not p r o t e c t the b a c t e r i u m f r o m phagocytosis or c o m p l e m e n t fixation [1]. T h e r e has been no a t t e m p t to establish a role for colanic acid in a d h e r e n c e or c o l o n i z a t i o n in the intestinal tract. H o w e v e r , its w i d e s p r e a d o c c u r r e n c e a m o n g m e m ber o f the E s c h e r i c h i a and S a l m o n e l l a g e n e r a indicates that it m u s t h a v e s o m e function. Since the natural habitat of these b a c t e r i a is the intestinal tract o f w a r m - b l o o d e d animals, it is reasonable to a s s u m e that colanic acid plays s o m e part in allowing survival and g r o w t h at this site. E P S is an excellent candidate for an adhesin. E P S m o l e c u l e s h a v e b e e n s h o w n to mediate or enh a n c e a d h e r e n c e o f different bacterial species to epithelial cells [6, 8, 15, 16, 19]. The regulation o f E P S p r o d u c t i o n b y strains 932 and 932P generally parallels w h a t had p r e v i o u s l y b e e n d e m o n s t r a t e d for adh e r e n t ability [10]. U n d e r m o s t culture conditions tested, strain 932P p r o d u c e d m o r e E P S than its parental strain 932, and it a d h e r e d better. P r o d u c t i o n o f E P S w a s e n h a n c e d at t e m p e r a t u r e s o f 30~ or less w h e n cells w e r e g r o w n aerobically, or at 37~ anaerobically, conditions u n d e r w h i c h a d h e r e n t ability is also e n h a n c e d . This study further d e m o n strated that high levels o f E P S expression are correlated with strong a d h e r e n t ability. The d e m o n s t r a tion that E P S is a m a j o r cell surface structure that strongly binds to L P S and proteins also s u p p o r t s s o m e a d h e r e n t potential. W h e t h e r the magnitude o f that effect is great e n o u g h that E P S could be considered the actual m e d i a t o r o f a d h e r e n c e is u n k n o w n . It a p p e a r s that the p r e s e n c e o f the plasmide n c o d e d fimbria is u n i m p o r t a n t for a d h e r e n c e o f strains that are e x p r e s s i n g E P S , b e c a u s e the plasm i d - c u r e d strain 932P a d h e r e s as well as or better

than its parental strain [10]. H o w e v e r , it is possible that a d h e r e n c e o f E H E C is not a simple o n e - a d h e s i n / o n e - r e c e p t o r p h e n o m e n o n . M a n y studies h a v e revealed that b a c t e r i a rarely rely o n a single adhesin. C o o p e r a t i o n b e t w e e n fimbriae and E P S in a d h e r e n c e has b e e n d e m o n s t r a t e d in bacterial p a t h o g e n s o f the respiratory, urinary, and intestinal tracts [6, 8, 16]. It is likely that a similar situation exists for the E H E C .

ACKNOWLEDGMENTS We thank Paul Lewandoski for excellent technical assistance. This project was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, and by contributions from the food industry. Structural analysis was supported in part by the USDA/DOE/NSF Plant Science Centers program; this particular center has been funded by the Department of Energy grant DE-FG09-87-ER13810.

Literature Cited 1. Allen PM, Fisher D, Saunders JR, Hart CA (1987) The role of capsular polysaccharide K21b of Klebsiella and of the structurally related colanic acid polysaccharide of Escherichia coli in resistance to phagocytosis and serum killing, J Med Microbiol 24:363-370 2. Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acid. Anal Biochem 54:484-489 3. Christensen GD, Simpson WA, Bisno AL, Beachey EH (1982) Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 37:318-326 4. CostertonJW, Irvin RT, ChengKJ (1981) The bacterialglycocalyx in nature and disease. Annu Rev Microbio135:299-324 5. Cross AS (1990) The biologic significance of bacterial encapsulation. Curr Topics Microbiol Immunol 150:87-95 6. Glorioso JC, Jones GW, Rush HG, Pentler LJ, Darif CA, Coward JE (1982) Adhesion of type A Pasteurella multocida to rabbit pharyngeal cells and its possible role in rabbit respiratory tract infections. Infect Immun 35:1103-1109 7. Grant WD, Sutherland IW, Wilkinson JF (1969) Exopolysaccharide colanic acid and its occurrence in the Enterobacteriaceae. J Bacteriol 100:1187-1193 8. Hadad JJ, Gyles CL (1982) The role of K antigens of enteropathogenic Escherichia coli in colonization of the small intestine of calves. Can J Comp Med 46:21-26 9. Host AH, Dankert J, Hulstaert CE, Feiger J (1986) Cell surface characteristics of coagulase-negative staphylococci and their adherence to fluorinated poly(ethylenepropylene). Infect Immun 51:294-301 10. Junkins AD, Doyle MP (1989) Comparison of adherence properties of Escherchia coli O157:H7 and a 60-megadalton plasmid-cured derivative. Curt Microbiol 19:21-27 11. Karch J, Heeseman J, Laufs R, O'Brien AD, Tackett CO, Levine MM (1987) A plasmid of enterohemorrhagic Escherichia coli O157:H7 is required for expression of a new timbrial antigen and for adhesion to epithelial cells. Infect lmmun 55:455-461 12. Laemmli UK (1970) Cleavage of structural proteins during

A.D. Junkins and M.P. Doyle: Exopolysaccharide Production by EHEC

13.

14.

15.

16.

17.

18.

19.

20. 21.

assembly of the head of the bacteriophage T4. Nature 227:680-685 Mackie EB, Brown KN, Lain J, Costerton JW (1979) Morphological stabilization of capsules of group B streptococci, types Ia, Ib, II, and III, with specific antibody. J Bacteriol 138:609-617 Maloney PC, Schneider H, Brandt BL (1972) Production and degradation of serogroup B Neisseria meningitidis polysaccharide. Infect Immun 6:657-661 Marcus H, Austria A, Baker NR (1989) Adherence of Pseudomonas aeruginosa to tracheal epithelium. Infect Immun 57:1050-1053 McLean RJC, Nickel JC, Noakes VC, Costerton JW (1985) An in vivo ultarstructural study on infectious kidney stone genesis. Infect Immun 49:805-811 Morck DW, Watts TC, Acres SD, Costerton JW (1988) Electron microscopic examination of cells of Pasteurella haernolytica-A1 in experimentally infected cattle. Can J Vet Res 52:343-348 Moxon ER, Kroll JS (1990) The role of bacterial polysaccharide capsules as virulence factors. Curr Topics Microbiol Immunol 150:65-85 Onderdonk AB, Moon NE, Kasper DL, Bartlett JG (1978) Adherence of Bacteroides fragilis in vivo. Infect Immun 19:1083-1087 Rose MC (1989) Characterization of human tracheobronchial mucin glycoproteins. Methods Enzymol 179:3-17 Sherman PM, Soni R (1988) Adherence of Vero cytotoxinproducing Escherichia coli of serotype O157:H7 to human

22.

23.

24. 25.

26.

27.

28.

29.

17

epithelial cells in tissue culture: role of outer membranes as bacterial adhesins. J Med Microbiol 26:11-17 Sherman P, Soni R, Petric M, Karmali K (1987) Surface properties of the Vero cytotoxin-producing Escherichia coli O157:H7. Infect Immun 55:1824-1829 Sutherland IW (1969) Structural studies on colanic acid, the common exopolysaccharide found in the Enterobacteriaceae, by partial acid hydrolysis. Oligosaccharides from colanic acid. Biochem J 115:935-945 Sutherland, IW (1971) Enzymic hydrolysis of colanic acid. Eur J Biochem 23:582-587 Tsai CM, Frasch CE (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115-119 Tzipori S, Gibson R, Montanaro J (1989) Nature and distribution of mucosal lesions associated with enteropathogenic and enterohemorrhagic Escherichia coli in piglets and the role of plasmid-mediated factors. Infect Immun 57:1142-1150 Wadolkowski EA, Burris JA, O'Brien AD (1990) Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7. Infect Immun 58:24382445 Wardi AH, Michos GA (1972) Alcian blue staining of glycoproteins in acrylamide disc electrophoresis. Anal Biochem 49:607-609 Yan YL (1990) A simple and inexpensive method for drying high-percentage polyacrylamide gradient gels. BioTechniques 8:381-382

Demonstration of exopolysaccharide production by enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli O157:H7 produces visibly slimy colonies when grown on Sorbitol/MacConkey or Maloney's agar plates at room temperatu...
2MB Sizes 0 Downloads 0 Views