896
Adherence Characteristics to Human Small Intestinal Mucosa of Escherichia coli Isolated from Patients with Diarrhea or Urinary Tract Infections Tatsuo Yamamoto, Kazuhiko Fujita, and Takeshi Yokota
From the Departments of Bacteriology and Urology, School of Medicine, Juntendo University, Tokyo, Japan
Adherence to mucosa is the initial, essential step for some strains of Escherichia coli to develop pathogenicity [1-8]. Enterotoxigenic E. coli (ETEC) isolated from patients with diarrhea produce pilus adhesins such as colonization factor antigen (CFA)/I or CFA/II [1, 6, 9]. With these pili, ETEC adhere to the villus epithelial cell surface (microvilli) of the human intestine [10]. In the case of uropathogenic E. coli (UPEC) isolated from patients with urinary tract infections, pilus adhesins such as type 1 pili play a role in adherence to urinary tract mucosa [11-13]. Human small intestinal mucosa has a mucus coating, villi, and lymphoid follicle epithelium at the mucosal surface. Human lymphoid follicle epithelium occasionally contains M cells [14]. M cells are morphologically characterized by microfolds at the luminal surface [14] and playa key role in initial intestinal local immune responses by causing transepithelial migration of bacterial antigens from the lumen into the lymphoid tissues [15]. By using formalin-fixed (or native) human small intestinal mucosa, we investigated adherence sites and levels of bacterial enteropathogens: Vibrio cholerae 01 [16, 17], V. cholerae non-Ol [18], Vibrio parahaemolyticus [19], and ETEC with CFA/I or CFA/II pili [20]. In such an in vitro adherence model system, V. cholerae 01, V.
Received 29 June 1989; revised 14 March 1990. Grant support: Ministry of Education, Science, and Culture of Japan. Reprints and correspondence: Dr. Tatsuo Yamamoto, Department of Bacteriology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyoku, Tokyo, Japan. The Journal of Infectious Diseases 1990;162:896-908 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6204-0017$01.00
cholerae non-Ol, and V. parahaemolyticus grown for 1"\.13 h at 37°C, unlike those grown for 1"\.120 h, showed strong adherence ability, hemagglutinating activity, and motility. Also, V. cholerae 01 and V. parahaemolyticus adhered more strongly to M cells (microfolds) than to other epithelial lymphoid follicle cells (microvilli) [21, 22]. Human urinary bladder mucosa contains transitional epithelium that consists of several layers of cells. The first layer (called the superficial layer) is in contact with urine in the bladder lumen and overlies the second (intermediate) layer. The superficial layer contains microridges at the luminal cell surface [23]. Previously we investigated adherence sites and levels for type 1 pili-possessing UPEC using formalin-fixed or untreated (native) human ureteral mucosa [24]. Here we report adherence experiments using human small intestinal mucosa or human urinary bladder mucosa and ETEC, E. coli fecal isolates, and UPEC. We also tested pilus adhesins for their ability to adhere to M cell microfolds.
Materials and Methods Bacteria, media, and bacterialgrowth. The bacterial strains used in this study are summarized in table 1. For bacterial growth, we used CFA agar [34], consisting of 1% casamino acids (Difco Laboratories, Detroit), 0.15 % yeast extract (Difco), 0.005 % MgS0 4 , 0.0005 % MnCh, and 2 % agar (pH 7.4), and brain-heart infusion (BHI) agar (Eiken Chemical, Tokyo)as the solid medium. Bacteria streaked on the agar's surface were incubated for 3-20 h at 37°C. When nutrient broth (Eiken) was used as the liquid medium, the incubation period was 48 h at 37°C without agitation [35, 36]. Hemagglutinin assay. To measure CFA/I pili-associated hemagglutinin (HA) activity [34], bacteria grown on the surface of CFA
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on December 12, 2014
Formalin-fixed human ileal mucosa and formalin-fixed or untreated (native) human urinary bladder mucosa were used to test the adherence ability of Escherichia coli enterotoxigenic (ETEC) or uropathogenic (UPEC) for humans. When grown on colonization factor antigen (CFA) agar for 3 h at 37°C, ETEC with CFA/I or CFA/II pili had typical peritrichous flagella and adhered strongly to human ileal lymphoid follicle and villus epithelium. In contrast, E. coli cells with CFA/I or CFA/II pili and possessing very weak or no motility displayed low levels of adherence to the epithelium. UPEC, which possessed type 1 pili and rarely had flagella, strongly adhered to human urinary bladder mucosa but not to human ileal epithelium. Type 1 pili-possessing E. coli isolated from human feces behaved as did UPEC. Moreover, M cells (microfolds) present in human ileal lymphoid follicle epithelium provided adherence sites for type 1 pili but not for CFA/I or CFA/II pili. These data demonstrate the importance of bacterial motility in efficient in vitro adherence to human ileal epithelia, in contrast to human urinary epithelia, and the adhesin specificity of bacterial adherence to M cell microfolds.
E. coli Adherence to Human Mucosa
JID 1990;162 (October)
Table 1.
897
Bacterial strains used in adherence study. Phenotype
Description, source or reference Clinical isolate of ETEC; [9, 25, 26], this study
H10407-P
CPA/I, type I, LT(Ih), ST(Ia), ST(Ib), 078:HII Type I, LT(Ih), ST(Ia), 078:H11
JE2217 JE2217 (pCSlt 1)
Fla-, PilCPA/I, ST(Ib), Kmr, Fla-
H10176 H10176 CPA/II-
CPA/II, LT(I), ST(I)
Strain Escherichia coli H10407
JN13 (pCSltl)
Type 1 Type 1
CPA/I, ST(Ib), Kmr
Strain that produces no detectable hemagglutinin and displays no detectable adherence; [27], this study JNI3 carrying pCSltl; [27]
NOTE. EPEC, enterotoxigenic E. coli; UPEC, uropathogenic E. coli; CFA/I, CFA/I pili; type 1, type 1 piIi; CFA/II, CFA/II pili; LT, heat-labile enterotoxin; ST, heat-stable enterotoxin; Kmr, kanamycin resistance; Fla", non-flagella-producing; Pil non-pili-producing. Subtypes of enterotoxins are indicated in parentheses. 078:HII, bacterial serotype. * Negative for D-mannose-resistant hemagglutinins (MRHA) with human (PI antigen-positive) erythrocytes. Adhesive pili such as P [28, 29, 30], S [31, 32], and M [30, 33] ofUPEC display this MRHA. No detectable P pili was confirmed by slide agglutination using a dry spot galnt-sgalp-larex agglutination kit (Orion Diagnostica, Espoo, Finland). r
or BHI agar plates were suspended in PBS (pH 7.4) to a concentration of 300 Klett units (measured in a Klett-Summerson photoelectric colorimeter with a red filter; Klett Manufacturing, New York). Twofold serial dilutions were then made with PBS, and 100-pl samples were mixed with 100 p,l of 3 % human (PI antigen-positive) erythrocytes, containing 1% (wt/vol) D-mannose, in a 24-well multidish plate (diameter of each well, 15 mm; A/S Nunc, Roskilde, Denmark). After the plates stood for 20-30 min at room temperature (f\J22°C), HA titers were determined with a light microscope (24-well plate method [16, 17]). To measure CFA/II pili-associated HA activity [37], bacterial cells grown on the surface of CFA agar plates were suspended to a concentration of 300 Klett units. HA titers were determined as described above,except that bovine erythrocytes and an incubation temperature of O°Cwere substituted. Tomeasure type 1 pili-associated HA activity [35], bacterial cells grown in nutrient broth were pelleted by centrifugation (at 3000 rpm for 20 min) and suspended in PBS to a concentration of 300 Klett units. HA titers were then determined as described above for CFA/I pili, except that guinea pig erythrocytes were. used instead of human erythrocytes and the addition of D-mannose was omitted. Preparation ofsmallintestinespecimens. Terminal ileum specimens from patients (aged 38 and 54 years) with ascending colon cancer were excised at Juntendo Hospital and prepared as described previously [16, 17]. The segments were immediately opened, and the mucosal side (from the muscularis mucosae to the mucosal epithelium with mucus) was saved and washed several times with cold (4°C) PBS. The mucosa was then fixed with 10% formalin in a modified Krebs-Ringer solution (KRT [38]) consisting of 7.5 g of NaCI, 0.383 g of KCI, 0.318 g of MgS04'7H20, and 0.305 g of CaCh/1 in 10 mM Tris-HCI (pH 7.4) and maintained at 4°C. Be-
,
fore adherence experiments, the formalin-fixed mucosa was cut into 0.5-cm 2 pieces and washed for 3 h with cold (4°C) KRT with two buffer changes (500 rn1 each). Rabbit ileal specimens were prepared as described by Nakasone and Iwanaga [39]. The ileum was excised from an adult rabbit (Japanese White) weighing 2.5 kg, washed with cold (4°C) PBS, and fixed with 10% formalin in KRT. The specimens were cut into pieces and washed as above. Preparation ofurinarybladderspecimens. Anterior wall human bladder specimens were taken during suprapubic prostatectomy from patients (aged 65 and 71 years) with benign prostatic hypertrophy; the patients had no urinary tract infections. A slice (0.5 cm-) of bladder mucosa was immediately used for adherence experiments. In some experiments, the bladder mucosa was fixed with 10% formalin in KRT and maintained at 4°C. Before adherence experiments, the formalin-fixed bladder mucosa was cut into 0.5-cm 2 pieces and washed for 3 h with cold (4°C) KRT with two buffer changes (500 rn1 each). Adherence test. Bacteria were grown on CFA or BHI agar for 3-20 h at 37°C or in nutrient broth for 48 h at 37°C. Adherence experiments were conducted as described previously [16-18, 20, 24]. Pieces of intestine or bladder were immersed in 1.5-2 rn1 of the bacterial suspension at 600 Klett units in PBS, followed by incubation for 10 min at 28°C. The samples were immediately washed four times in PBS, fixed in a KRT solution containing 2.5 % (vol/vol)glutaraldehyde and 2 % (wt/vol) tannic acid for 2 h at room temperature, and subsequently postfixed in 1% (wt/vol) osmium tetroxide for 2 h (or overnight) at 4°C. Scanning electron microscopy. Fixed intestine or bladder samples were dehydrated with acetone and critical-point-dried. The sam-
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E15, EI6 B7, B8 Citrobacter freundii JN13
H10407 derivative lacking a CPA/I-, STIbcoding plasmid pCS I; [9, 25, 26] [27] JE2217 carrying a pCSI-derivative plasmid pCSltl labeled with Km" transposon Tn5; [25,27] Clinical isolate of ETEC; D. G. Evans, [20] H10176 derivative lacking a CPA/II-, LTI-, STI-coding plasmid; D. G. Evans, [20] Clinical isolates* of UPEC; this study, [24] Isolates* from feces of a healthy human; this study
898
Yamamoto et al.
termined by indirect ELISA (competitive antigen modification) [43] using the purified flagella sample and antiserum to HII flagella; also a dot-immunobinding assay [44] using a nitrocellulose filter (pore size, 0.45 /-1m ; Bio-Rad Laboratories, Richmond, CA), an ImmunBlot assay kit (Bio-Rad), and antiserum to Hll flagella, was done as previously described [27].
Results
Adherence of CFA/Il pili-possessing ETEC. CFA/II pili-possessing ETEC strain H10176 grown for 3 h adhered extensively to human ileal villus epithelium and lymphoid follicle epithelium (figure lA, B). The lymphoid follicle epithelium was a better adherence target than was the villus epithelium. In marked contrast, H10176 cells grown for 20 h showed much lower adherence (table 2). Adherent cells grown for 3 h possessed typical peritrichous flagella (figure IC) while those grown for 20 h rarely possessed flagella (figure
Figure 1. Adherence of CFA/II pili-possessing E. coli strain HI0176, grown for 3 h (A-C) or 20 h (D), to formalin-fixed human ileal villi (A) or lymphoid follicle epithelium (B-D) . Arrows in C indicate flagella. Bars: A, B, 25 /-1m ; C, D, 1 ~m.
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pies were then coated with gold-palladium and examined by scanning electron microscopy. In experiments in which adherence levels were determined, 30 observation fields (magnification x4000; 23 X 28 ~m) were randomly chosen and photographed and the number of bacteria recorded . The average number of bacteria per electron microscopic field constituted the adherence index. Serologic assay offlagella . To quantify flagella antigen production, ETEC strain 65 (serotype 013:Kll:HIl) was grown overnight in BHI broth (Eiken), and harvested bacteria were suspended in saline containing 0.1% HCHO. Flagella were removed from the cells by homogenization with a Woding blender and purified essentially as described [40]. Rabbit antiserum to these flagella samples was prepared as described [41]. Totest Hll flagella antigen production, bacteria were suspended in PBS and sonicated in a Branson sonicator (model 200; Branson Sonic Power, Danbury, CT) for 90 s at 50 W. Cell debris was removed by centrifugation for 10 min at 10,000 g, and the protein concentration of the supernatant was determined by dye-binding assay [42] . Amounts of Hll flagella antigen in the supernatant were de-
JID 1990;162 (October)
JIO 1990;162 (October)
E. coli Adherence to Human Mucosa
899
Table 2. Adherence of bacteria to the epithelial surfaces of formalin-fixed ileal villi and lymphoid follicles: marked bacterial adherence to the human ileal epithelium with CF A pili and role of flagella. Adherence index for
Strain, medium
Human ileal epithelium Lymphoid follicles
± 15.0 ± 5.5 ± 0.3
Villi
± 9.5 ± 3.6 ± 0.6
0.2 0.1 0.1
O±O ± 0.5 ± 0.4
0.3 0.2
3 20 48
35.5 6.0 0.1
3 20 48
0.2 0.3
± 0.7
0.2 0.1
3 20 48
11.3 3.7 6.3
± 8.9 ± 2.8 ± 5.6
5.0 1.8 2.8
± 7.2 ± 1.7 ± 5.6
3 20 48
0.2 ± 0.2 ± 6.4 ± (0.9 ±
0.1
± 0.4
JE2217 (pCSltl) CFA agar CFA agar BHI agar BHI agar Related strains of Citrobacter freundii JN13 (pCSltl) CFA agar CFA agar JN13 CFA agar CFA agar Uropathogenic E. coli E16 CFA agar CFA agar Nutrient broth
3 20 48
Fecal isolate E. coli B8 CFA agar CFA agar Nutrient broth
3 20 48
3 20 3 20
± 0.6 NO
0.5 0.5 4.8 1.4)*
o ± 0.2 o±0 0.1
± 0.4 NO
9.5 3.4 0.2
o ± 0.2 5:6 (0.8
± 6.0 ± 1.4)*
± 11.2 ± 5.7
16.2 7.4
± 6.3 ± 3.7
3 20
0.5 0.4
± 1.0 ± 0.6
0.2
± 0.4
NO 1.0
± 0.9
NO NO 0.2 ± 0.7
O±O 0.2 ± 0.5 1.1 ± 1.6
O±O ± 0.4 ± 8.7
0.1 7.4
O±O
25.6 8.5
o ± 0.2 o ± 0.2 0.4 ± 0.6 3.0 ± 5.5 (0 ± 0)* o±0 ± 0.6 o±0
0.3
± 0.9 ± 0.6 ± 0.4
O±O ± 0.9 ± 0.7
NO NO NO NO
0.8 ± 1.7 0.2 ± 0.5 o ± 0.2
3 20
o±0
Rabbit ileal villus epithelium
0.3 0.2
± 0.8 ± 0.5
o ± 0.2 o ± 0.2 0.2 ± 0.6 2.6 ± 2.5 13.9 ± 17.1 (0.3 ± 0.7)*
3.0 1.7 8.7
± 3.2 ± 2.8 ± 8.1
NOTE. Data are mean ± SD for 30 determinations. ND = not done. * Adherence index determined in the presence of 1 % (wt/vol) D-mannose.
ID). CFA/II pili-associated HA activity was much more obvious with 20 h-grown than with 3 h-grown cells (table 3). Bacterial cell-bound CFA/II pili that attached to the microvilli at the pilus-free side were seen with both 3 h- and 20 h-grown cells (figure IC, D). Human ileal lymphoid follicle epithelium consisted mainly of cells with microvilli and occasional M cells with microfolds at the luminal surface. HI0176 cells grown on CFA agar for
3 h at 37°C (thus possessing typical peritrichous flagella and CFA/II pili and manifesting a high adherence ability to microvilli) would not adhere to M cell microfolds (figure 2). Cells that adhered to microvilli all possessed. peritrichous flagella. HI0176 cells grown for 20 h also failed to adhere to M cell microfolds. Similarly, E. coli HI0176 without CFA/II pili grown on CFA agar for 3 h at 37°C possessed typical peritrichous flagella,
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Enterotoxigenic Escherichia coli H10176 CFA agar CFA agar Nutrient broth H10176 CFA/IICFA agar CFA agar Nutrient broth H10407 CFA agar CFA agar Nutrient broth HI0407-P CFA agar CFA agar Nutrient broth
Length (h) of incubation
Yamamoto et al.
900
110 1990;162 (October)
Table 3. Pilus-associated hemagglutinin (HA) levels of bacterial strains.
Strain, medium
HA titer of MRHA (bovine)
MSHA (guinea pig)
Type of pili (HA) detected
3 20 48
Adherence Characteristics to Human Small Intestinal Mucosa of Escherichia coli Isolated from Patients with Diarrhea or Urinary Tract Infections Tatsuo Yamamoto, Kazuhiko Fujita, and Takeshi Yokota
From the Departments of Bacteriology and Urology, School of Medicine, Juntendo University, Tokyo, Japan
Adherence to mucosa is the initial, essential step for some strains of Escherichia coli to develop pathogenicity [1-8]. Enterotoxigenic E. coli (ETEC) isolated from patients with diarrhea produce pilus adhesins such as colonization factor antigen (CFA)/I or CFA/II [1, 6, 9]. With these pili, ETEC adhere to the villus epithelial cell surface (microvilli) of the human intestine [10]. In the case of uropathogenic E. coli (UPEC) isolated from patients with urinary tract infections, pilus adhesins such as type 1 pili play a role in adherence to urinary tract mucosa [11-13]. Human small intestinal mucosa has a mucus coating, villi, and lymphoid follicle epithelium at the mucosal surface. Human lymphoid follicle epithelium occasionally contains M cells [14]. M cells are morphologically characterized by microfolds at the luminal surface [14] and playa key role in initial intestinal local immune responses by causing transepithelial migration of bacterial antigens from the lumen into the lymphoid tissues [15]. By using formalin-fixed (or native) human small intestinal mucosa, we investigated adherence sites and levels of bacterial enteropathogens: Vibrio cholerae 01 [16, 17], V. cholerae non-Ol [18], Vibrio parahaemolyticus [19], and ETEC with CFA/I or CFA/II pili [20]. In such an in vitro adherence model system, V. cholerae 01, V.
Received 29 June 1989; revised 14 March 1990. Grant support: Ministry of Education, Science, and Culture of Japan. Reprints and correspondence: Dr. Tatsuo Yamamoto, Department of Bacteriology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyoku, Tokyo, Japan. The Journal of Infectious Diseases 1990;162:896-908 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6204-0017$01.00
cholerae non-Ol, and V. parahaemolyticus grown for 1"\.13 h at 37°C, unlike those grown for 1"\.120 h, showed strong adherence ability, hemagglutinating activity, and motility. Also, V. cholerae 01 and V. parahaemolyticus adhered more strongly to M cells (microfolds) than to other epithelial lymphoid follicle cells (microvilli) [21, 22]. Human urinary bladder mucosa contains transitional epithelium that consists of several layers of cells. The first layer (called the superficial layer) is in contact with urine in the bladder lumen and overlies the second (intermediate) layer. The superficial layer contains microridges at the luminal cell surface [23]. Previously we investigated adherence sites and levels for type 1 pili-possessing UPEC using formalin-fixed or untreated (native) human ureteral mucosa [24]. Here we report adherence experiments using human small intestinal mucosa or human urinary bladder mucosa and ETEC, E. coli fecal isolates, and UPEC. We also tested pilus adhesins for their ability to adhere to M cell microfolds.
Materials and Methods Bacteria, media, and bacterialgrowth. The bacterial strains used in this study are summarized in table 1. For bacterial growth, we used CFA agar [34], consisting of 1% casamino acids (Difco Laboratories, Detroit), 0.15 % yeast extract (Difco), 0.005 % MgS0 4 , 0.0005 % MnCh, and 2 % agar (pH 7.4), and brain-heart infusion (BHI) agar (Eiken Chemical, Tokyo)as the solid medium. Bacteria streaked on the agar's surface were incubated for 3-20 h at 37°C. When nutrient broth (Eiken) was used as the liquid medium, the incubation period was 48 h at 37°C without agitation [35, 36]. Hemagglutinin assay. To measure CFA/I pili-associated hemagglutinin (HA) activity [34], bacteria grown on the surface of CFA
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on December 12, 2014
Formalin-fixed human ileal mucosa and formalin-fixed or untreated (native) human urinary bladder mucosa were used to test the adherence ability of Escherichia coli enterotoxigenic (ETEC) or uropathogenic (UPEC) for humans. When grown on colonization factor antigen (CFA) agar for 3 h at 37°C, ETEC with CFA/I or CFA/II pili had typical peritrichous flagella and adhered strongly to human ileal lymphoid follicle and villus epithelium. In contrast, E. coli cells with CFA/I or CFA/II pili and possessing very weak or no motility displayed low levels of adherence to the epithelium. UPEC, which possessed type 1 pili and rarely had flagella, strongly adhered to human urinary bladder mucosa but not to human ileal epithelium. Type 1 pili-possessing E. coli isolated from human feces behaved as did UPEC. Moreover, M cells (microfolds) present in human ileal lymphoid follicle epithelium provided adherence sites for type 1 pili but not for CFA/I or CFA/II pili. These data demonstrate the importance of bacterial motility in efficient in vitro adherence to human ileal epithelia, in contrast to human urinary epithelia, and the adhesin specificity of bacterial adherence to M cell microfolds.
E. coli Adherence to Human Mucosa
JID 1990;162 (October)
Table 1.
897
Bacterial strains used in adherence study. Phenotype
Description, source or reference Clinical isolate of ETEC; [9, 25, 26], this study
H10407-P
CPA/I, type I, LT(Ih), ST(Ia), ST(Ib), 078:HII Type I, LT(Ih), ST(Ia), 078:H11
JE2217 JE2217 (pCSlt 1)
Fla-, PilCPA/I, ST(Ib), Kmr, Fla-
H10176 H10176 CPA/II-
CPA/II, LT(I), ST(I)
Strain Escherichia coli H10407
JN13 (pCSltl)
Type 1 Type 1
CPA/I, ST(Ib), Kmr
Strain that produces no detectable hemagglutinin and displays no detectable adherence; [27], this study JNI3 carrying pCSltl; [27]
NOTE. EPEC, enterotoxigenic E. coli; UPEC, uropathogenic E. coli; CFA/I, CFA/I pili; type 1, type 1 piIi; CFA/II, CFA/II pili; LT, heat-labile enterotoxin; ST, heat-stable enterotoxin; Kmr, kanamycin resistance; Fla", non-flagella-producing; Pil non-pili-producing. Subtypes of enterotoxins are indicated in parentheses. 078:HII, bacterial serotype. * Negative for D-mannose-resistant hemagglutinins (MRHA) with human (PI antigen-positive) erythrocytes. Adhesive pili such as P [28, 29, 30], S [31, 32], and M [30, 33] ofUPEC display this MRHA. No detectable P pili was confirmed by slide agglutination using a dry spot galnt-sgalp-larex agglutination kit (Orion Diagnostica, Espoo, Finland). r
or BHI agar plates were suspended in PBS (pH 7.4) to a concentration of 300 Klett units (measured in a Klett-Summerson photoelectric colorimeter with a red filter; Klett Manufacturing, New York). Twofold serial dilutions were then made with PBS, and 100-pl samples were mixed with 100 p,l of 3 % human (PI antigen-positive) erythrocytes, containing 1% (wt/vol) D-mannose, in a 24-well multidish plate (diameter of each well, 15 mm; A/S Nunc, Roskilde, Denmark). After the plates stood for 20-30 min at room temperature (f\J22°C), HA titers were determined with a light microscope (24-well plate method [16, 17]). To measure CFA/II pili-associated HA activity [37], bacterial cells grown on the surface of CFA agar plates were suspended to a concentration of 300 Klett units. HA titers were determined as described above,except that bovine erythrocytes and an incubation temperature of O°Cwere substituted. Tomeasure type 1 pili-associated HA activity [35], bacterial cells grown in nutrient broth were pelleted by centrifugation (at 3000 rpm for 20 min) and suspended in PBS to a concentration of 300 Klett units. HA titers were then determined as described above for CFA/I pili, except that guinea pig erythrocytes were. used instead of human erythrocytes and the addition of D-mannose was omitted. Preparation ofsmallintestinespecimens. Terminal ileum specimens from patients (aged 38 and 54 years) with ascending colon cancer were excised at Juntendo Hospital and prepared as described previously [16, 17]. The segments were immediately opened, and the mucosal side (from the muscularis mucosae to the mucosal epithelium with mucus) was saved and washed several times with cold (4°C) PBS. The mucosa was then fixed with 10% formalin in a modified Krebs-Ringer solution (KRT [38]) consisting of 7.5 g of NaCI, 0.383 g of KCI, 0.318 g of MgS04'7H20, and 0.305 g of CaCh/1 in 10 mM Tris-HCI (pH 7.4) and maintained at 4°C. Be-
,
fore adherence experiments, the formalin-fixed mucosa was cut into 0.5-cm 2 pieces and washed for 3 h with cold (4°C) KRT with two buffer changes (500 rn1 each). Rabbit ileal specimens were prepared as described by Nakasone and Iwanaga [39]. The ileum was excised from an adult rabbit (Japanese White) weighing 2.5 kg, washed with cold (4°C) PBS, and fixed with 10% formalin in KRT. The specimens were cut into pieces and washed as above. Preparation ofurinarybladderspecimens. Anterior wall human bladder specimens were taken during suprapubic prostatectomy from patients (aged 65 and 71 years) with benign prostatic hypertrophy; the patients had no urinary tract infections. A slice (0.5 cm-) of bladder mucosa was immediately used for adherence experiments. In some experiments, the bladder mucosa was fixed with 10% formalin in KRT and maintained at 4°C. Before adherence experiments, the formalin-fixed bladder mucosa was cut into 0.5-cm 2 pieces and washed for 3 h with cold (4°C) KRT with two buffer changes (500 rn1 each). Adherence test. Bacteria were grown on CFA or BHI agar for 3-20 h at 37°C or in nutrient broth for 48 h at 37°C. Adherence experiments were conducted as described previously [16-18, 20, 24]. Pieces of intestine or bladder were immersed in 1.5-2 rn1 of the bacterial suspension at 600 Klett units in PBS, followed by incubation for 10 min at 28°C. The samples were immediately washed four times in PBS, fixed in a KRT solution containing 2.5 % (vol/vol)glutaraldehyde and 2 % (wt/vol) tannic acid for 2 h at room temperature, and subsequently postfixed in 1% (wt/vol) osmium tetroxide for 2 h (or overnight) at 4°C. Scanning electron microscopy. Fixed intestine or bladder samples were dehydrated with acetone and critical-point-dried. The sam-
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on December 12, 2014
E15, EI6 B7, B8 Citrobacter freundii JN13
H10407 derivative lacking a CPA/I-, STIbcoding plasmid pCS I; [9, 25, 26] [27] JE2217 carrying a pCSI-derivative plasmid pCSltl labeled with Km" transposon Tn5; [25,27] Clinical isolate of ETEC; D. G. Evans, [20] H10176 derivative lacking a CPA/II-, LTI-, STI-coding plasmid; D. G. Evans, [20] Clinical isolates* of UPEC; this study, [24] Isolates* from feces of a healthy human; this study
898
Yamamoto et al.
termined by indirect ELISA (competitive antigen modification) [43] using the purified flagella sample and antiserum to HII flagella; also a dot-immunobinding assay [44] using a nitrocellulose filter (pore size, 0.45 /-1m ; Bio-Rad Laboratories, Richmond, CA), an ImmunBlot assay kit (Bio-Rad), and antiserum to Hll flagella, was done as previously described [27].
Results
Adherence of CFA/Il pili-possessing ETEC. CFA/II pili-possessing ETEC strain H10176 grown for 3 h adhered extensively to human ileal villus epithelium and lymphoid follicle epithelium (figure lA, B). The lymphoid follicle epithelium was a better adherence target than was the villus epithelium. In marked contrast, H10176 cells grown for 20 h showed much lower adherence (table 2). Adherent cells grown for 3 h possessed typical peritrichous flagella (figure IC) while those grown for 20 h rarely possessed flagella (figure
Figure 1. Adherence of CFA/II pili-possessing E. coli strain HI0176, grown for 3 h (A-C) or 20 h (D), to formalin-fixed human ileal villi (A) or lymphoid follicle epithelium (B-D) . Arrows in C indicate flagella. Bars: A, B, 25 /-1m ; C, D, 1 ~m.
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pies were then coated with gold-palladium and examined by scanning electron microscopy. In experiments in which adherence levels were determined, 30 observation fields (magnification x4000; 23 X 28 ~m) were randomly chosen and photographed and the number of bacteria recorded . The average number of bacteria per electron microscopic field constituted the adherence index. Serologic assay offlagella . To quantify flagella antigen production, ETEC strain 65 (serotype 013:Kll:HIl) was grown overnight in BHI broth (Eiken), and harvested bacteria were suspended in saline containing 0.1% HCHO. Flagella were removed from the cells by homogenization with a Woding blender and purified essentially as described [40]. Rabbit antiserum to these flagella samples was prepared as described [41]. Totest Hll flagella antigen production, bacteria were suspended in PBS and sonicated in a Branson sonicator (model 200; Branson Sonic Power, Danbury, CT) for 90 s at 50 W. Cell debris was removed by centrifugation for 10 min at 10,000 g, and the protein concentration of the supernatant was determined by dye-binding assay [42] . Amounts of Hll flagella antigen in the supernatant were de-
JID 1990;162 (October)
JIO 1990;162 (October)
E. coli Adherence to Human Mucosa
899
Table 2. Adherence of bacteria to the epithelial surfaces of formalin-fixed ileal villi and lymphoid follicles: marked bacterial adherence to the human ileal epithelium with CF A pili and role of flagella. Adherence index for
Strain, medium
Human ileal epithelium Lymphoid follicles
± 15.0 ± 5.5 ± 0.3
Villi
± 9.5 ± 3.6 ± 0.6
0.2 0.1 0.1
O±O ± 0.5 ± 0.4
0.3 0.2
3 20 48
35.5 6.0 0.1
3 20 48
0.2 0.3
± 0.7
0.2 0.1
3 20 48
11.3 3.7 6.3
± 8.9 ± 2.8 ± 5.6
5.0 1.8 2.8
± 7.2 ± 1.7 ± 5.6
3 20 48
0.2 ± 0.2 ± 6.4 ± (0.9 ±
0.1
± 0.4
JE2217 (pCSltl) CFA agar CFA agar BHI agar BHI agar Related strains of Citrobacter freundii JN13 (pCSltl) CFA agar CFA agar JN13 CFA agar CFA agar Uropathogenic E. coli E16 CFA agar CFA agar Nutrient broth
3 20 48
Fecal isolate E. coli B8 CFA agar CFA agar Nutrient broth
3 20 48
3 20 3 20
± 0.6 NO
0.5 0.5 4.8 1.4)*
o ± 0.2 o±0 0.1
± 0.4 NO
9.5 3.4 0.2
o ± 0.2 5:6 (0.8
± 6.0 ± 1.4)*
± 11.2 ± 5.7
16.2 7.4
± 6.3 ± 3.7
3 20
0.5 0.4
± 1.0 ± 0.6
0.2
± 0.4
NO 1.0
± 0.9
NO NO 0.2 ± 0.7
O±O 0.2 ± 0.5 1.1 ± 1.6
O±O ± 0.4 ± 8.7
0.1 7.4
O±O
25.6 8.5
o ± 0.2 o ± 0.2 0.4 ± 0.6 3.0 ± 5.5 (0 ± 0)* o±0 ± 0.6 o±0
0.3
± 0.9 ± 0.6 ± 0.4
O±O ± 0.9 ± 0.7
NO NO NO NO
0.8 ± 1.7 0.2 ± 0.5 o ± 0.2
3 20
o±0
Rabbit ileal villus epithelium
0.3 0.2
± 0.8 ± 0.5
o ± 0.2 o ± 0.2 0.2 ± 0.6 2.6 ± 2.5 13.9 ± 17.1 (0.3 ± 0.7)*
3.0 1.7 8.7
± 3.2 ± 2.8 ± 8.1
NOTE. Data are mean ± SD for 30 determinations. ND = not done. * Adherence index determined in the presence of 1 % (wt/vol) D-mannose.
ID). CFA/II pili-associated HA activity was much more obvious with 20 h-grown than with 3 h-grown cells (table 3). Bacterial cell-bound CFA/II pili that attached to the microvilli at the pilus-free side were seen with both 3 h- and 20 h-grown cells (figure IC, D). Human ileal lymphoid follicle epithelium consisted mainly of cells with microvilli and occasional M cells with microfolds at the luminal surface. HI0176 cells grown on CFA agar for
3 h at 37°C (thus possessing typical peritrichous flagella and CFA/II pili and manifesting a high adherence ability to microvilli) would not adhere to M cell microfolds (figure 2). Cells that adhered to microvilli all possessed. peritrichous flagella. HI0176 cells grown for 20 h also failed to adhere to M cell microfolds. Similarly, E. coli HI0176 without CFA/II pili grown on CFA agar for 3 h at 37°C possessed typical peritrichous flagella,
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on December 12, 2014
Enterotoxigenic Escherichia coli H10176 CFA agar CFA agar Nutrient broth H10176 CFA/IICFA agar CFA agar Nutrient broth H10407 CFA agar CFA agar Nutrient broth HI0407-P CFA agar CFA agar Nutrient broth
Length (h) of incubation
Yamamoto et al.
900
110 1990;162 (October)
Table 3. Pilus-associated hemagglutinin (HA) levels of bacterial strains.
Strain, medium
HA titer of MRHA (bovine)
MSHA (guinea pig)
Type of pili (HA) detected
3 20 48
