Vol. 26, No. 2
INFECTION AND IMMUNITY, Nov. 1979, p. 736-743 0019-9567/79/11-0736/08$02.00/0
Quantitation of the Adherence of an Enteropathogenic Escherichia coli to Isolated Rabbit Intestinal Brush Borders CHRISTOPHER P. CHENEY,* EDGAR C. BOEDEKER, AND SAMUEL B. FORMAL Departments of Gastroenterology and Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012 Received for publication 17 August 1979
Two assays were developed to quantitate the adherence of an Escherichia coli strain (RDEC-1) known to colonize the mucosal surface of the small intestine of rabbits to brush borders isolated from rabbit intestinal epithelial cells. In the first assay, the mean adherence per rabbit brush border was determined by counting the number of organisms adhering to each of 40 brush borders under phase microscopy. The mean adherence of RDEC-1 (11.5 ± 0.7 per rabbit brush border) was significantly greater than adherence of two nonpathogenic strains: HS (2.7 ± 0.4 per rabbit brush border) and 640 (0.8 ± 0.1 per rabbit brush border). A similar distinction between the adherence of RDEC-1 and the control (nonadherent) organisms could be made more rapidly by determining the percentage of the total number of brush borders which had 10 or more adherent organisms; this second assay was used to define the optimum conditions for adherence. Maximum adherence was seen within 15 min. Adherence was temperature dependent, with adherence after 1 min at 37°C being fourfold greater than that at 4°C. The pH optimum for adherence was between 6.5 and 7.0, and adherence was abolished below pH 5.0. With the first, more sensitive assay, the effect of electrolytes and a number of hexoses and hexosamines on adherence was analyzed. RDEC-1 adherence was inhibited at high ionic strengths; however, adherence was not influenced at moderately high concentrations (20 mg/mil) by either D-mannose or L-fucose, in contrast to the case for other reported enteric pathogens. These two quantitative in vitro assays for adherence produce consistent results and have been used to partially characterize the adherence of RDEC-1 to rabbit brush borders.
Adherence of enteropathogenic bacteria to the mucosal surface of intestinal epithelial cells is an important determinant of virulence for some enteric organisms. Adherence of pathogenic Escherichia coli appears to facilitate the ability of the organism to colonize the small bowel by allowing the pathogens to replicate while resisting the possible clearing action of intestinal peristalsis. Enteric organisms already included among this group of adherent pathogens are porcine K88 (19, 22, 28, 38) and bovine K99 (4) strains of E. coli, Vibrio cholerae (20, 21), and possibly the human enterotoxigenic E. coli, H10407 (12, 34). Mucosal adherence is not an exclusive property of enteric infections, but has also been described with gonococcal (30) and E. coli (10, 23) infections of the urogenital tract and with streptococcal infections of the oral cavity (2) and heart valves (17). In most of these cases, bacterial adherence results from the mutual recognition and interaction of surface structures from both the bacteria and the host cells. Recent investigations
have succeeded in identifying some of the adhesins on the surface of pathogenic organisms (12, 13, 18, 19, 28, 30, 31, 34) and to a lesser extent in identifying the constituents of the receptor on the host epithelial cells (1, 15, 29; C. P. Cheney, E. C. Boedeker, and S. B. Formal, Fed. Proc. 37:1727, 1978). To study the optimal conditions for adherence and to further probe the biochemical nature of the involved structures by studying the effect of inhibitors on this interaction, an in vitro adherence model is desirable. A suitable in vitro model should correlate with the known characteristics of the infective process in vivo and in particular should possess the same host and tissue specificity. For our studies in adherence, we chose the rabbit model of diarrhea reported by Cantey and Blake (5). They described a fatal diarrhea in young rabbits caused by a piliated enteropathogenic E. coli, RDEC-1 (015:NM). RDEC-1 appears to be an unusual organism among virulent enteric E. coli strains in that it is a noninvasive organism which does not synthesize either a
736
VOL. 26, 1979
ADHERENCE TO RABBIT BRUSH BORDERS
classical heat-stable or heat-labile E. coli toxin. This organism was found to adhere and multiply on the surface of the ileum and the colon (36). Furthermore, this organism has also been shown to bind to and cause agglutination of ileal brush borders and microvillus membranes isolated from rabbit ileal epithelial cells but not to similar membranes prepared from rat, guinea pig, or human tissue (E. C. Boedeker, Y. K. Higgins, S. B. Formal, and A. Takeuchi, Gastroenterology 72:1154, 1977) and not to cells isolated from other rabbit tissue (bladder and erythrocytes). Thus, the RDEC-1 rabbit diarrheal model is both convenient and highly appropriate for the in vitro study of bacterial diarrhea associated with species- and tissue-specific mucosal adherence. The present studies were conducted to develop sensitive in vitro assays for the quantitation of bacterial adherence which in turn were utilized for determining the optimal conditions for adherence and for testing the influence of ionic strength on this process. In addition, we tested the influence on RDEC-1 adherence of several hexoses and hexosamines, including Dmannose and L-fucose, which have been shown to inhibit mucosal adherence in other bacterial systems.
MATERIALS AND METHODS Brush border preparation. Male New Zealand White rabbits weighing between 1 and 3 kg were anesthetized by intravenous injection of pentobarbital (65 mg/kg). A 50-cm segment of distal ileum was excised and immediately rinsed with cold (40C) ethylenediaminetetraacetic acid (EDTA) buffer (5 mM EDTA, 5 mM Na2H2PO4, pH 7.5). All further procedures were done at 40C. The ileum was opened, the mucosal surface was scraped with glass slides, and the scrapings were placed in EDTA buffer. Rabbit brush borders (RBB) were prepared from these mucosal scrapings by the method of Donaldson et al. (8) by differential centrifugation. Purity of the brush border preparations was assessed by examination under phase microscopy (Fig. 1A) and by following the enrichment of maltase activity by the method of Dahlquist (7). Brush border preparations were subjected to repeated slow-speed centrifugation until nuclear contamination was essentially eliminated. Maltase activity of the final rabbit ileal brush borders was enriched between 9- and 13-fold compared with the homogenates. The final brush border fraction was resuspended in phosphatebuffered saline (PBS) (0.145 M NaCl, 0.01 M Na2HPO4-NaH2PO4, pH 7.0). Brush borders were prepared fresh daily for each assay. Protein was estimated by the method of Lowry et al. (26), and brush borders were diluted to a final concentration of 1 mg/ml with PBS. The number of RBB per milligram of brush border protein was determined by counting in a hemacytometer under phase microscopy and found to average 2.65 (±0.25) x 107 RBB/mg.
737
Bacterial strains. Three strains of E. coli were examined for their ability to adhere to rabbit brush borders. The RDEC-1 strain (015:K?:NM) was isolated by J. R. Cantey (Veterans Administration Hospital, Charleston, S.C.) from laboratory rabbits that spontaneously developed diarrhea. Two strains of E. coli were used as controls: 640 (06:H5), isolated from the intestinal flora of a normal rabbit, and HS (O undetermined:H4), isolated from a normal human fecal sample and used earlier as a nonpathogenic control strain (25). Neither strain agglutinated RBB in vitro or caused diarrheal disease in rabbits in vivo (C. P. Cheney, E. C. Boedeker, M. K. Diadato, and S. B. Formal, Gastroenterology 76:1112, 1979). Bacterial cultures were grown overnight at 37°C in 10 ml of Penassay broth (Difco Laboratories, Detroit, Mich.) in screw-topped culture tubes. Bacteria from a single tube were pelleted at 2,400 x g at 23°C for 5 min, washed twice in PBS, and resuspended in 2 mil of PBS. Final concentrations of the bacteria were measured by plating serial 10-fold dilutions of the PBS suspensions on MacConkey agar plates and determining colony-forming units per milliliter after overnight incubation at 37°C. The final concentrations of bacteria used in this study were 2 x 109 to 4 x 109/ml. In vitro bacterial adherence assays. A 50-p1 amount of purified RBB (50 ytg of protein, 106 brush borders) was mixed with a 20-,ul suspension of bacteria (108 E. coli) and 30,l of PBS and allowed to incubate for up to 30 min with shaking at 23°C. The tubes were examined visually for agglutination, and phase microscopic examination at 600x revealed large aggregates of coagglutinated brush borders and RDEC-1 (Fig. 1B). Initially this reaction was evaluated by rating the aggregates from 0 to +4, but such scoring was not sufficiently quantitative. However, it was found that mixing for 10 s on setting no. 5 with a Vortex-Genie (Scientific Industries Inc., Bohemia, N.Y.) would disperse the large aggregates into individual brush borders without eliminating the brush border-bacterial interactions, thus permitting quantitation of the number of organisms adhering to each brush border. In the first assay (assay I), bacterial adherence to dispersed brush borders was quantitated by observation under phase microscopy at a magnification of 600x and reported as the number of organisms adhering per RBB. To more rapidly monitor bacterial adherence, a second adherence assay (assay II) was established with the same incubation conditions. Adherence in this assay was expressed as the percentage of RBB possessing 10 or more adherent E. coli within a given area (0.4 mm2) of a hemacytometer. An average of 200 RBB were counted in this assay. Effect of ionic strength. RBB (50 td), RDEC-1 (20 il), and portions (30 til) from stock solutions of varying concentrations of either KCl, NaCl, or KH2PO4 made up in PBS were mixed together and incubated for 15 min at 23°C. In assay I, RDEC-1 adherence to RBB was quantitated at a range of final concentrations of KCl (30 to 600 mM) and at a single final concentration of 600 mM for NaCl and KH2PO4 in at least five separate brush border preparations. The data were normalized to control values for RDEC1 adherence in PBS alone, and the results were expressed as percent of control adherence +1 standard
738
INFECT. IMMUN.
CHENEY, BOEDEKER, AND FORMAL
error. The reaction mixture was maintained at pH 7.0, and the osmolality of the solutions at their highest ionic strength was between 1,192 and 1,300 mosM as determined with a Digimatic-Osmometer (model 3D, Advanced Instruments Inc., Needham, Mass.). Carbohydrate inhibition. Portions (30 jil) from standard stock solutions (66 mg/mi of PBS) of the hexoses and hexosamines D-mannose, yeast mannan, a-methyl-mannoside, L-fucose, D-glucose, D-galactose,
N-acetyl-D-galactosamine, or N-acetyl-D-glucosamine were added to 50 pl of RBB (50 jtg of protein) and 20 td of RDEC-1. The final carbohydrate concentration was 20 mg/ml or approximately 125 mM for each hexose. The mixture was incubated for 15 min, and the average number of adherent RDEC-1 on 40 RBB was determined (assay I). The data were normalized to control values for RDEC-1 adherence in the absence of carbohydrate, and the results were expressed as percentage of control adherence +1 standard error.
RESULTS Microscopic appearance of E. coli interactions with isolated RBB. When the three test strains of E. coli, RDEC-1, 640, and HS, were incubated with isolated rabbit intestinal brush borders, visual agglutination was noted within 15 min with RDEC-1 but not with either of the control strains, HS or 640. At this stage, large aggregates of coagglutinated RDEC-1 and brush borders were observed under phase mi-
F
c-
AI
C
X
croscopy (Figure 1B), whereas 640 remained uniformly dispersed, and only occasional clumping of up to four brush borders with HS could be found. After blending in a Vortex mixer, the large aggregates of RDEC-1 and brush borders were dispersed, and individual brush borders with adherent RDEC-1 were observed (Fig. 10). The cytoplasmic side of the brush borders was essentially free of any adherent RDEC-1 organisms as far as could be detected by phase microscopy at this magnification. In each RDEC-1 incubation, a number of brush borders remained free of adherent RDEC-1 organisms. Strain 640 continued to show an absence of adherence to RBB even after mixing (Fig. iD). Quantitation of in vitro RDEC-1 adherence to RBB. Assay I: mean number of adherent organisms per RBB. To demonstrate quantitatively that the adherence of RDEC-1 was significantly greater than that of the control E. coli strains, the number of adherent organisms on each of 40 brush borders after a 15-min incubation at 230C was counted in at least three separate studies (different brush border preparations) per strain. From these data, the mean number of adherent organisms per brush border standardd error) was calculated. Consistent with the microscopic appearance,
N.
elli 4g. ,~
..e :f:.
il
.:P4-
..!
.."
:,ff
AP
! 4
4
S
5
~. 4~~~ ^
FIG. 1. Phase-contrast photomicrographs (x600) of the reactions of RBB with RDEC-1 or 640 E. coli after 15 min of incubation at 230C. (A) Control RBB preparation without added organisms. (B) Aggregates of coagglutinated RDEC-1 and RBB. (C) Vortex dispersion of RDEC-1 and RBB aggregates resulting in solitary RBB containing adherent RDEC-1. (D) Nonadherent control strain of E. coli (640) with RBB.
ADHERENCE TO RABBIT BRUSH BORDERS
VOL. 26, 1979
RDEC-1 adherence was significantly greater than that of control strains with an average of 11.5 ± 0.7 RDEC-1 bound per brush
mean
border as compared with a mean 2.7 ± 0.4 HS brush border (P < 0.001) and 0.8 ± 0.1 640 per brush border (P < 0.001). Complete enumeration of adherence in a single tube required approximately 5 to 7 min and therefore severely limited the number of reactions which could be studied in a single day. Assay II: percentage of RBB with 10 adherent E. coli. The enumeration data from the above studies were tabulated to display the numerical distribution of RBB which had the following ranges of adherent E. coli per RBB: 0, 1 to 4, 5 to 9, 10 to 14, 15 to 19, 20 to 24, and greater than 24 (Table 1). Analysis of the data in this table reveals that 55% (22/40) of the RBB possessed 10 or more adherent E. coli when incubated with RDEC-1 as compared with 5% (2/40) when incubated with HS or 0% (0/40) when incubated with 640. Therefore, determining the percentage of RBB with 10 or more adherent organisms within a given area of the hemacytometer (0.4 mm2) provided an assay which was sensitive enough to distinguish between strongly adherent and either weakly adherent or nonadherent E. coli strains and yet could be performed in approximately 2 min per sample or in one-third the time of the previous assay. Assay II was subsequently used as our rapid method of quantitation of RDEC-1 adherper
739
incubation was continued up to 30 min. The 640 strain failed to adhere throughout the entire time span studied. (ii) Effect of temperature. Incubations performed at 4, 23, and 370C demonstrated that RDEC-1 adherence is temperature dependent (Fig. 3). Maximal adherence at 370C (82.6 ± 2.0%) was observed at 15 min. This degree of adherence was not achieved at 40C within 15 min; however, continued incubation to 60 min revealed that adherence at 40C had reached a level comparable to that seen at 370C after 15 min. The half time for maximal adherence was less than 1 min at 370C, 2 min at 230C, and 7 min at 40C. As shown in Fig. 3, binding within the first minute was fourfold greater at 370C than at 40C (P < 0.001). 100-
w
ax
co
80-
0
RDEC
a
I
- O
60-
0
40co
ws:n
a:
c
cr n c
20640
ence.
T T l 1 Characteristics of RDEC-1 adherence to 5 15 10 rabbit leal brush borders. (i) Time course. TIME (min) Observations at 1, 5, and 15 min with assay II FIG. 2. Time profile of bacterial adherence to RBB revealed that binding of RDEC-1 occurs rapidly at 230C. Adherence was monitored under phase miat 230C and reaches a plateau (71.6 ± 3.5% of croscopy by determining the percentage of brush borRBB with 210 adherent organisms) within 15 ders containing 10 or more adherent bacteria. Each min (Fig. 2). A slight drop in adherence of 7% point represents the mean of four experiments done (not shown on the graph) was observed when in duplicate standard error. 0
I
±
TABLE 1. Distribution of rabbit ileal brush borders with varying numbers of adherent E. coli Adherent E. coli per brush border
0 1-4
5-9 10-14
RDEC-1a
No. of brush borders with adherent E. coli (7)b HS (4) 640 (3)
2.3 ± 0.9c (5.7)d 6.2 ± 1.0 (15.5) 8.7 ± 1.3 (22.0) 9.2 ± 1.2 (23.0) 8.2 ± 1.1 (9.5) 3.8 + 0.7 (9.5) 1.5 ± 0.6 (4.0)
16.8 ± 3.4 (42.0) 15.5 ± 1.4 (38.8) 5.5 ± 2.5 (14) 1.8 ± 1.4 (4) 0.5 ± 0.5 (1) 0 0 40.0
15-19 20-24
INFECTION AND IMMUNITY, Nov. 1979, p. 736-743 0019-9567/79/11-0736/08$02.00/0
Quantitation of the Adherence of an Enteropathogenic Escherichia coli to Isolated Rabbit Intestinal Brush Borders CHRISTOPHER P. CHENEY,* EDGAR C. BOEDEKER, AND SAMUEL B. FORMAL Departments of Gastroenterology and Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012 Received for publication 17 August 1979
Two assays were developed to quantitate the adherence of an Escherichia coli strain (RDEC-1) known to colonize the mucosal surface of the small intestine of rabbits to brush borders isolated from rabbit intestinal epithelial cells. In the first assay, the mean adherence per rabbit brush border was determined by counting the number of organisms adhering to each of 40 brush borders under phase microscopy. The mean adherence of RDEC-1 (11.5 ± 0.7 per rabbit brush border) was significantly greater than adherence of two nonpathogenic strains: HS (2.7 ± 0.4 per rabbit brush border) and 640 (0.8 ± 0.1 per rabbit brush border). A similar distinction between the adherence of RDEC-1 and the control (nonadherent) organisms could be made more rapidly by determining the percentage of the total number of brush borders which had 10 or more adherent organisms; this second assay was used to define the optimum conditions for adherence. Maximum adherence was seen within 15 min. Adherence was temperature dependent, with adherence after 1 min at 37°C being fourfold greater than that at 4°C. The pH optimum for adherence was between 6.5 and 7.0, and adherence was abolished below pH 5.0. With the first, more sensitive assay, the effect of electrolytes and a number of hexoses and hexosamines on adherence was analyzed. RDEC-1 adherence was inhibited at high ionic strengths; however, adherence was not influenced at moderately high concentrations (20 mg/mil) by either D-mannose or L-fucose, in contrast to the case for other reported enteric pathogens. These two quantitative in vitro assays for adherence produce consistent results and have been used to partially characterize the adherence of RDEC-1 to rabbit brush borders.
Adherence of enteropathogenic bacteria to the mucosal surface of intestinal epithelial cells is an important determinant of virulence for some enteric organisms. Adherence of pathogenic Escherichia coli appears to facilitate the ability of the organism to colonize the small bowel by allowing the pathogens to replicate while resisting the possible clearing action of intestinal peristalsis. Enteric organisms already included among this group of adherent pathogens are porcine K88 (19, 22, 28, 38) and bovine K99 (4) strains of E. coli, Vibrio cholerae (20, 21), and possibly the human enterotoxigenic E. coli, H10407 (12, 34). Mucosal adherence is not an exclusive property of enteric infections, but has also been described with gonococcal (30) and E. coli (10, 23) infections of the urogenital tract and with streptococcal infections of the oral cavity (2) and heart valves (17). In most of these cases, bacterial adherence results from the mutual recognition and interaction of surface structures from both the bacteria and the host cells. Recent investigations
have succeeded in identifying some of the adhesins on the surface of pathogenic organisms (12, 13, 18, 19, 28, 30, 31, 34) and to a lesser extent in identifying the constituents of the receptor on the host epithelial cells (1, 15, 29; C. P. Cheney, E. C. Boedeker, and S. B. Formal, Fed. Proc. 37:1727, 1978). To study the optimal conditions for adherence and to further probe the biochemical nature of the involved structures by studying the effect of inhibitors on this interaction, an in vitro adherence model is desirable. A suitable in vitro model should correlate with the known characteristics of the infective process in vivo and in particular should possess the same host and tissue specificity. For our studies in adherence, we chose the rabbit model of diarrhea reported by Cantey and Blake (5). They described a fatal diarrhea in young rabbits caused by a piliated enteropathogenic E. coli, RDEC-1 (015:NM). RDEC-1 appears to be an unusual organism among virulent enteric E. coli strains in that it is a noninvasive organism which does not synthesize either a
736
VOL. 26, 1979
ADHERENCE TO RABBIT BRUSH BORDERS
classical heat-stable or heat-labile E. coli toxin. This organism was found to adhere and multiply on the surface of the ileum and the colon (36). Furthermore, this organism has also been shown to bind to and cause agglutination of ileal brush borders and microvillus membranes isolated from rabbit ileal epithelial cells but not to similar membranes prepared from rat, guinea pig, or human tissue (E. C. Boedeker, Y. K. Higgins, S. B. Formal, and A. Takeuchi, Gastroenterology 72:1154, 1977) and not to cells isolated from other rabbit tissue (bladder and erythrocytes). Thus, the RDEC-1 rabbit diarrheal model is both convenient and highly appropriate for the in vitro study of bacterial diarrhea associated with species- and tissue-specific mucosal adherence. The present studies were conducted to develop sensitive in vitro assays for the quantitation of bacterial adherence which in turn were utilized for determining the optimal conditions for adherence and for testing the influence of ionic strength on this process. In addition, we tested the influence on RDEC-1 adherence of several hexoses and hexosamines, including Dmannose and L-fucose, which have been shown to inhibit mucosal adherence in other bacterial systems.
MATERIALS AND METHODS Brush border preparation. Male New Zealand White rabbits weighing between 1 and 3 kg were anesthetized by intravenous injection of pentobarbital (65 mg/kg). A 50-cm segment of distal ileum was excised and immediately rinsed with cold (40C) ethylenediaminetetraacetic acid (EDTA) buffer (5 mM EDTA, 5 mM Na2H2PO4, pH 7.5). All further procedures were done at 40C. The ileum was opened, the mucosal surface was scraped with glass slides, and the scrapings were placed in EDTA buffer. Rabbit brush borders (RBB) were prepared from these mucosal scrapings by the method of Donaldson et al. (8) by differential centrifugation. Purity of the brush border preparations was assessed by examination under phase microscopy (Fig. 1A) and by following the enrichment of maltase activity by the method of Dahlquist (7). Brush border preparations were subjected to repeated slow-speed centrifugation until nuclear contamination was essentially eliminated. Maltase activity of the final rabbit ileal brush borders was enriched between 9- and 13-fold compared with the homogenates. The final brush border fraction was resuspended in phosphatebuffered saline (PBS) (0.145 M NaCl, 0.01 M Na2HPO4-NaH2PO4, pH 7.0). Brush borders were prepared fresh daily for each assay. Protein was estimated by the method of Lowry et al. (26), and brush borders were diluted to a final concentration of 1 mg/ml with PBS. The number of RBB per milligram of brush border protein was determined by counting in a hemacytometer under phase microscopy and found to average 2.65 (±0.25) x 107 RBB/mg.
737
Bacterial strains. Three strains of E. coli were examined for their ability to adhere to rabbit brush borders. The RDEC-1 strain (015:K?:NM) was isolated by J. R. Cantey (Veterans Administration Hospital, Charleston, S.C.) from laboratory rabbits that spontaneously developed diarrhea. Two strains of E. coli were used as controls: 640 (06:H5), isolated from the intestinal flora of a normal rabbit, and HS (O undetermined:H4), isolated from a normal human fecal sample and used earlier as a nonpathogenic control strain (25). Neither strain agglutinated RBB in vitro or caused diarrheal disease in rabbits in vivo (C. P. Cheney, E. C. Boedeker, M. K. Diadato, and S. B. Formal, Gastroenterology 76:1112, 1979). Bacterial cultures were grown overnight at 37°C in 10 ml of Penassay broth (Difco Laboratories, Detroit, Mich.) in screw-topped culture tubes. Bacteria from a single tube were pelleted at 2,400 x g at 23°C for 5 min, washed twice in PBS, and resuspended in 2 mil of PBS. Final concentrations of the bacteria were measured by plating serial 10-fold dilutions of the PBS suspensions on MacConkey agar plates and determining colony-forming units per milliliter after overnight incubation at 37°C. The final concentrations of bacteria used in this study were 2 x 109 to 4 x 109/ml. In vitro bacterial adherence assays. A 50-p1 amount of purified RBB (50 ytg of protein, 106 brush borders) was mixed with a 20-,ul suspension of bacteria (108 E. coli) and 30,l of PBS and allowed to incubate for up to 30 min with shaking at 23°C. The tubes were examined visually for agglutination, and phase microscopic examination at 600x revealed large aggregates of coagglutinated brush borders and RDEC-1 (Fig. 1B). Initially this reaction was evaluated by rating the aggregates from 0 to +4, but such scoring was not sufficiently quantitative. However, it was found that mixing for 10 s on setting no. 5 with a Vortex-Genie (Scientific Industries Inc., Bohemia, N.Y.) would disperse the large aggregates into individual brush borders without eliminating the brush border-bacterial interactions, thus permitting quantitation of the number of organisms adhering to each brush border. In the first assay (assay I), bacterial adherence to dispersed brush borders was quantitated by observation under phase microscopy at a magnification of 600x and reported as the number of organisms adhering per RBB. To more rapidly monitor bacterial adherence, a second adherence assay (assay II) was established with the same incubation conditions. Adherence in this assay was expressed as the percentage of RBB possessing 10 or more adherent E. coli within a given area (0.4 mm2) of a hemacytometer. An average of 200 RBB were counted in this assay. Effect of ionic strength. RBB (50 td), RDEC-1 (20 il), and portions (30 til) from stock solutions of varying concentrations of either KCl, NaCl, or KH2PO4 made up in PBS were mixed together and incubated for 15 min at 23°C. In assay I, RDEC-1 adherence to RBB was quantitated at a range of final concentrations of KCl (30 to 600 mM) and at a single final concentration of 600 mM for NaCl and KH2PO4 in at least five separate brush border preparations. The data were normalized to control values for RDEC1 adherence in PBS alone, and the results were expressed as percent of control adherence +1 standard
738
INFECT. IMMUN.
CHENEY, BOEDEKER, AND FORMAL
error. The reaction mixture was maintained at pH 7.0, and the osmolality of the solutions at their highest ionic strength was between 1,192 and 1,300 mosM as determined with a Digimatic-Osmometer (model 3D, Advanced Instruments Inc., Needham, Mass.). Carbohydrate inhibition. Portions (30 jil) from standard stock solutions (66 mg/mi of PBS) of the hexoses and hexosamines D-mannose, yeast mannan, a-methyl-mannoside, L-fucose, D-glucose, D-galactose,
N-acetyl-D-galactosamine, or N-acetyl-D-glucosamine were added to 50 pl of RBB (50 jtg of protein) and 20 td of RDEC-1. The final carbohydrate concentration was 20 mg/ml or approximately 125 mM for each hexose. The mixture was incubated for 15 min, and the average number of adherent RDEC-1 on 40 RBB was determined (assay I). The data were normalized to control values for RDEC-1 adherence in the absence of carbohydrate, and the results were expressed as percentage of control adherence +1 standard error.
RESULTS Microscopic appearance of E. coli interactions with isolated RBB. When the three test strains of E. coli, RDEC-1, 640, and HS, were incubated with isolated rabbit intestinal brush borders, visual agglutination was noted within 15 min with RDEC-1 but not with either of the control strains, HS or 640. At this stage, large aggregates of coagglutinated RDEC-1 and brush borders were observed under phase mi-
F
c-
AI
C
X
croscopy (Figure 1B), whereas 640 remained uniformly dispersed, and only occasional clumping of up to four brush borders with HS could be found. After blending in a Vortex mixer, the large aggregates of RDEC-1 and brush borders were dispersed, and individual brush borders with adherent RDEC-1 were observed (Fig. 10). The cytoplasmic side of the brush borders was essentially free of any adherent RDEC-1 organisms as far as could be detected by phase microscopy at this magnification. In each RDEC-1 incubation, a number of brush borders remained free of adherent RDEC-1 organisms. Strain 640 continued to show an absence of adherence to RBB even after mixing (Fig. iD). Quantitation of in vitro RDEC-1 adherence to RBB. Assay I: mean number of adherent organisms per RBB. To demonstrate quantitatively that the adherence of RDEC-1 was significantly greater than that of the control E. coli strains, the number of adherent organisms on each of 40 brush borders after a 15-min incubation at 230C was counted in at least three separate studies (different brush border preparations) per strain. From these data, the mean number of adherent organisms per brush border standardd error) was calculated. Consistent with the microscopic appearance,
N.
elli 4g. ,~
..e :f:.
il
.:P4-
..!
.."
:,ff
AP
! 4
4
S
5
~. 4~~~ ^
FIG. 1. Phase-contrast photomicrographs (x600) of the reactions of RBB with RDEC-1 or 640 E. coli after 15 min of incubation at 230C. (A) Control RBB preparation without added organisms. (B) Aggregates of coagglutinated RDEC-1 and RBB. (C) Vortex dispersion of RDEC-1 and RBB aggregates resulting in solitary RBB containing adherent RDEC-1. (D) Nonadherent control strain of E. coli (640) with RBB.
ADHERENCE TO RABBIT BRUSH BORDERS
VOL. 26, 1979
RDEC-1 adherence was significantly greater than that of control strains with an average of 11.5 ± 0.7 RDEC-1 bound per brush
mean
border as compared with a mean 2.7 ± 0.4 HS brush border (P < 0.001) and 0.8 ± 0.1 640 per brush border (P < 0.001). Complete enumeration of adherence in a single tube required approximately 5 to 7 min and therefore severely limited the number of reactions which could be studied in a single day. Assay II: percentage of RBB with 10 adherent E. coli. The enumeration data from the above studies were tabulated to display the numerical distribution of RBB which had the following ranges of adherent E. coli per RBB: 0, 1 to 4, 5 to 9, 10 to 14, 15 to 19, 20 to 24, and greater than 24 (Table 1). Analysis of the data in this table reveals that 55% (22/40) of the RBB possessed 10 or more adherent E. coli when incubated with RDEC-1 as compared with 5% (2/40) when incubated with HS or 0% (0/40) when incubated with 640. Therefore, determining the percentage of RBB with 10 or more adherent organisms within a given area of the hemacytometer (0.4 mm2) provided an assay which was sensitive enough to distinguish between strongly adherent and either weakly adherent or nonadherent E. coli strains and yet could be performed in approximately 2 min per sample or in one-third the time of the previous assay. Assay II was subsequently used as our rapid method of quantitation of RDEC-1 adherper
739
incubation was continued up to 30 min. The 640 strain failed to adhere throughout the entire time span studied. (ii) Effect of temperature. Incubations performed at 4, 23, and 370C demonstrated that RDEC-1 adherence is temperature dependent (Fig. 3). Maximal adherence at 370C (82.6 ± 2.0%) was observed at 15 min. This degree of adherence was not achieved at 40C within 15 min; however, continued incubation to 60 min revealed that adherence at 40C had reached a level comparable to that seen at 370C after 15 min. The half time for maximal adherence was less than 1 min at 370C, 2 min at 230C, and 7 min at 40C. As shown in Fig. 3, binding within the first minute was fourfold greater at 370C than at 40C (P < 0.001). 100-
w
ax
co
80-
0
RDEC
a
I
- O
60-
0
40co
ws:n
a:
c
cr n c
20640
ence.
T T l 1 Characteristics of RDEC-1 adherence to 5 15 10 rabbit leal brush borders. (i) Time course. TIME (min) Observations at 1, 5, and 15 min with assay II FIG. 2. Time profile of bacterial adherence to RBB revealed that binding of RDEC-1 occurs rapidly at 230C. Adherence was monitored under phase miat 230C and reaches a plateau (71.6 ± 3.5% of croscopy by determining the percentage of brush borRBB with 210 adherent organisms) within 15 ders containing 10 or more adherent bacteria. Each min (Fig. 2). A slight drop in adherence of 7% point represents the mean of four experiments done (not shown on the graph) was observed when in duplicate standard error. 0
I
±
TABLE 1. Distribution of rabbit ileal brush borders with varying numbers of adherent E. coli Adherent E. coli per brush border
0 1-4
5-9 10-14
RDEC-1a
No. of brush borders with adherent E. coli (7)b HS (4) 640 (3)
2.3 ± 0.9c (5.7)d 6.2 ± 1.0 (15.5) 8.7 ± 1.3 (22.0) 9.2 ± 1.2 (23.0) 8.2 ± 1.1 (9.5) 3.8 + 0.7 (9.5) 1.5 ± 0.6 (4.0)
16.8 ± 3.4 (42.0) 15.5 ± 1.4 (38.8) 5.5 ± 2.5 (14) 1.8 ± 1.4 (4) 0.5 ± 0.5 (1) 0 0 40.0
15-19 20-24
