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Chronic diarrhea in infants caused by adherent enteropathogenic Escherichia coli
358
March 1982 TheJournalofPEDIATRICS
Chronic diarrhea in infants caused by adherent enteropathogenic Escherichia coli Enteropathogenic Escherichia coli (Ol l l:K58:H-) was isolated from the small bowel of two infants with chronic diarrhea. Small bowel biopsy revealed focal adherence of the bacteria to the epithelium, accompanied by inflammation of the tissue. The isolates also adhered to tissue culture cells in densely packed aggregates. The pattern o f adherence to tissue culture cells were found only among strains of serogroups 0111 and 0119 when 196 enteric E. coil isolates were tested. The pathogenic mechanism of enteropathogenic E. coil thus appears related to dense colonization of intestinal tissue. Adherent strains may be detected by tissue culture assay.
Carla R. Clausen, Ph.D.,* and Dennis L. Christie, M.D., Seattle, Wash.
ESCHERICHIA COL! m a y produce diarrhea by two welldefined mechanisms: Shigella-like penetration of intestinal epithelium, or elaboration of one or more enterotoxins. ~-4 T h e m e c h a n i s m responsible for infantile diarrhea associated with certain serotypes of E. coli (enteropathogenic E. coli) has not been established. A l t h o u g h some E P E C isolates have been found to produce enterotoxins or invade epithelial cells, 5-7 the majority of stains are neither toxigenic nor invasive. 8 A n o t h e r potential m e c h a n i s m was suggested by the isolation of an E. coli (0125 a t : H 2 1 ) from the small intestine of an infant with enterocolitis. 9 Electron microscopy of the duodenal biopsy specimen from the infant demonstrated bacilli adherent to the epithelium, with disruption of the microvilli. No enterotoxins nor invasive properties were associated with the isolate. The pathology of the infant's disease was similar to t h a t produced by a strain of E. coli which causes diarrhea in rabbits and adheres in large numbers to r a b b i t intestinal epithelium. ~~This strain did not produce any of the known enterotoxins and did not appear to invade the epithelial cells. In our hospital, E P E C 0111 was recently isolated from the small bowel of two infants with chronic diarrhea. T h e isolates adhered to tissue culture cells in densely packed From the Gastroenterology Division, Children's Orthopedic Hospital and Medical Center, *Reprint address: Department of Laboratories, COHMC, ~ P.O. Box C-5371, 4800 Sand Point Way N.E.; Seattle, WA 98105.
Vol. 100, No. 3, pp. 358-361
aggregates. Small bowel biopsy revealed acute inflammatory changes and focal adherence by large numbers of E P E C 0111. Abbreviations used EPEC: enteropathogenic Escherichia coli COHMC: Children's Orthopedic Hospital and Medical Center CASE REPORTS
Patients. Two infants from the ,same central Washington community were referred to Children's Orthopedic Hospital and Medical Center in Seattle for diarrhea which had been present for longer than two weeks. The first patient, 5.5 months of age, was admitted 8/12/80 following three weeks of persistent diarrhea and vomiting. The infant had been admitted to another hospital on two occasions because of dehydration. He continued to have 10 to 12 liquid stools per day and failed to tolerate clear liquids or soy or casein hydrolysate formulas. On admission to COHMC he was found to have a weight less than the third percentile, and a serum albumin concentration of 3 gm/dl. His daily stool output averaged 400 ml while fasting. He could not maintain his body weight and total parenteral nutrition was necessary. The diarrhea began to resolve 48 hours after intravenous gentamicin therapy was started. He was discharged to be fed a casein hydrolysate formula 30 days after admission; his weight gain was 1 kg. The second patient was 15 months of age when admitted 9/23/80, more than two weeks after the onset of diarrhea, vomiting, and low-grade fever. She had been admitted previously to another hospital because of dehydration. Prior to admission to COHMC, she had lost 1 kg in weight, Her serum albumin
0022-3476/82/030358+04500.40/0 9 1982 The C. V. Mosby Co.
Volume 100 Number 3
Diarrhea caused by adherent E. coli
359
concentration was 2.4 gm/dl. Stool output while fasting averaged 300 ml daily. She was given total parenteral nutrition to maintain body weight. The diarrhea began to resolve five days after initiation of intravenous gentamicin therapy. The patient was discharged 18 days after admission, receiving soy formula and a nonlactose-containing diet. Weight gain during hospitalization was 1.3 kg. Small bowel biopsy, performed on both patients the day after admission, was submitted for histologic examination. Intestinal fluid obtained at the same time was submitted for culture. METHODS Tissue culture assay. Leighton tubes containing No. 1 coverslips were seeded with cells of a continuous human epithelial cell line (Hep II). The cells were grown in Eagle minimal essential medium supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100 gg/ml streptomycin. When monolayers were nearly confluent, the medium was replaced with medium not containing antibiotics or serum. The monolayers were inoculated with 0.2 ml of an 18-hour bacterial culture grown in Trypticase soy broth. After a two-hour incubation at 35~ the medium was replaced with fresh medium. The cell culture was incubated an additional three to four hours. The coverslips were then removed from the tubes, washed vigorously in phosphatebuffered saline (pH 7.0), and stained by a Wright-Giemsa technique. Fluorescent antibody method. Sections of deparaffinated and acetone-fixed biopsy tissue were stained with dilutions of 0111:K58 sera prepared in rabbits (BBL Microbiology Systems, Cockeysville, Md.) Duplicate sections were treated with heterologous EPEC sera to provide negative controls. After incubation at 25~ for 30 minutes with the primary antisera, the sections were washed in phosphatebuffered saline, dipped into tap water, and blotted dry. All sections were then stained with a 1:200 dilution of fluorescein conjugated goat anti-rabbit globulin (Antibodies Incorporated, Davis, Calif.). After a 30-minute incubation, the sections were washed as before, dried, and mounted with buffered glycerol. RESULTS Culture isolates. Cultures of intestinal fluid from both infants yielded an abundant growth of non-motile E. coll. No anaerobes were isolated. Serotyping was performed by the methods described by Edwards and Ewing. ~ Heated suspensions of the isolates were agglutinated by a greater than 1:1,000 dilution of 0111:K58 antiserum. The 0111:K58:H- serotype was isolated from subsequent stool specimens from both infants. No other enteric pathogens, including Salmonella, Shigella, Yersinia enterocolitica, Campylobacter fetus subspecies jejuni, or rotavirus were detected. Polymorphonuclear leukocytes were observed in
Fig. 1. Section of small bowel biopsy stained with rabbit anti0111 sera and fluorescein-conjugatedanti-rabbit globulin. the stool from the second patient but not in stools from the first. Blood cultures were sterile. Neither isolate was found to contain genes which encode for heat labile or heat stable enterotoxin by DNA homology studies) z* Both were resistant to ampicillin, carbenicillin, cephalothin, kanamycin, sulfisoxazole, and neomycin, but susceptible to chloramphenieol, gentamicin, and tobramycin by disk diffusion assay. Cultures obtained when the biopsy was repeated two weeks later did not yield the EPEC. Small bowel biopsy. The small intestinal biopsies of the two patients showed a severe mucosal lesion. The lamina propria was edematous and densely populated with neutrophils, macrophages, and lymphocytes. No trophozoites of Giardia lamblia were found. Large numbers of bacilli were observed adhering to the epithelium, but no invasion into the lamina propria was evident. The bacteria were not found diffusely throughout the mucosa, but in discrete areas sometimes extending into the crypts. Fluorescent antibody staining identified the adherent bacilli as EPEC 0111 (Fig. 1). The repeat biopsy specimens showed *DNA homologystudieswere performedby S. k. Moseley,Department of Microbiologyand Immunology,Universityof Washington,Seattle, Washington.
360
Clausen and Christie
The Journal of Pediatrics March 1982 from the two infants in the present study plus an isolate from a third patient with chronic diarrhea. This patient (2.5 months of age) was referred to COHMC in June, 1980; EPEC 0 l l 1 was isolated from stool only and not from the small bowel. However, the biopsy was performed two weeks following a short course of ampicillin and gentamicin. Isolates from these three patients had the same multi-resistant antibiogram, and the three infants came to COHMC from the same community. DISCUSSION
Fig. 2. Aggregates of bacilli adhering to Hep II tissue culture cells. Wright-Giemsa stain.
improvement in villus architecture and an absence of bacteria. Tissue culture assay. The attachment of the EPEC 0111 isolates to tissue culture (Fig. 2) was not confluent but in aggregates, giving the appearance of forming microcolonies. Cytoplasmic invasion of cells, which can be demonstrated in the assay by virulent strains of Shigella, was not apparent. During the past several years, a number of enteric Strains of E. coli isolated from infants and children with diarrheal disease have been examined for adherence by the tissue culture assay. The isolates were selected from stool cultures that did not yield bacterial pathogens, i.e., Salmonella, Shigella, Y. enterocolitica, or C. fetus ss jejuni. Only 11 of 196 strains adhered to Hep II cells as described for the intestinal fluid isolates. Nonadherent strains were removed from the monolayer by the washing procedure, so that only an occasional bacterium was observed in the stained preparation. Six of the positive strains were serotype 011 l:K58:H-. The other five were motile strains which agglutinated in 0119:K69 antiserum. The H (flagellar) antigen was n(~t determined. The six positive 0111 isolates include those
Enteropathogenic E. coli is not a homogeneous group of pathogens, since some strains of 026:H 11 J3 and 0128:H126 have been shown to elaborate enterotoxins, and strains of serogroup 028 are capable of Shigella-like invasion of cells, 7 The pathogenic mechanism for the majority of EPEC remains obscure. For example, two strains (0142:H6 and 0127:H6) isolated during outbreaks of infantile diarrhea produced a diarrheal illness in adult volunteers. Tests for heat-labile and heat-stable enterotoxin, as well as for invasiveness, were negative?4 However, there is some indication that these two isolates may induce fluid secretion from the bowel by another as yet uncharacterized toxin? 5 The present study reinforces evidence that infection with certain strains of EPEC may result in serious diarrhea; the pathogenesis appears related to tissue adherence. Adherence by strains of serogroup 0111 has been noted in several studies. By immunofluorescence, Drucker et a116 found EPEC attached to intestinal tissue in sections obtained at autopsy of 17 infants who had died of enteritis; 14 of the 17 were serogroup 0111. All of the EPEC-associated fatal cases had inflammatory changes in the intestine ranging from moderate (chronic inflammation with flattened villi) to severe (acute inflammation and mucosal necrosis). Polotsky et al ~3 illustrated by electron microscopy the attachment of some strains of EPEC, including 0111, to rabbit intestine. Rothbaum et a117 recently described an outbreak of infantile diarrhea caused by 0119:B 14(K69); the organism was isolated from both stool and duodenal aspirates. Bacteria were seen adhering in clusters to the mucosal surfaces of atrophic villi in small bowel biopsy tissue. Some strains of 0111 and 0119 possess virulence factors which enable them to colonize intestinal epithelial cells. Although a heretofore unrecognized toxin may mediate the subsequent diarrheal illness, the pathology noted in our patients does not correlate with the toxigenic disease, which leaves the bowel intact. Disruption of villi and inflammatory changes may be caused solely by the effect of firm attachment of the organism, with subsequent exposure of the host tissue to enzymatic activity and
Volume 1O0 Number 3
metabolic products of the a d h e r e n t bacteria. Dense colonization could conceivably interfere with normal absorption and with secretory processes of the intestinal epithelium. Finally, the a d h e r e n t strains can be detected in tissue culture assay. T h e p a t t e r n of adherence is unique and was found in only 11 of 196 enteric isolates of E. coli. All of the a d h e r e n t E. coli were in serogroups 0111 and 0119. T h e six 0111 isolates which adhered to Hep II cells were nonmotile, thus representing a serotype commonly associated with infantile d i a r r h e a (0111 :K58:H-). T h e a d h e r e n t 0119 strains were not completely characterized since ttagellar antigen typing was not performed. Thus it is not known if these isolates were 0119:K69:H6, which is the only serotype of the serogroup 0119 recognized as an E P E C . TM T h e assay reflected in vivo adherence to intestinal t i s s u e o f the two infants in the present study, and m a y serve as a tool to detect virulent strains of enteric E. coll. REFERENCES 1. Du Pont HL, Formal SB, Hornick RB, Snyder M J, Libonati JP, Sheahan DG, Le Brec EH, and Kalas JP: Pathogenesis of Escherichia coli diarrhea, N Engl J Med 285:1, 1971. 2. Sack RB, Gorbach SL, Banwell JG, Jacobs B, Chatterjee BD, and Mitra RC: Enterotoxigenic Escherichia coil isolated from patients with severe cholera-like disease. J Infect Dis 123:378, 1971. 3. Gorbach SL, Kean BH, Evans DG, Evans D J, and Bessudo D: Travelers' diarrhea and toxigenic Escherichia coli, N Engl J Med 292:933, 1975. 4. Sack DA, Merson MH, Wells JG, Sack RB, and Morris GK: Diarrhoea associated with heat-stable enterotoxin-producing strains of Escherichia coli, Lancet 2:239, 1975. 5. Gorbach SL, and Khurana CM: Toxigenic Escherichia coil: A cause of infantile diarrhea in Chicago, N Engl J Med 287:79t, 1972. 6. Reis MH, Castro AF, Toledo RM, and Trabulsi LR: Production of heat-stable enterotoxin by the 0128 serogroup of Escherichia coli, Infect Immun 24:289, 1979.
Diarrhea caused by adherent E. coli
36 1
7. Gemski P Jr, and Formal SB: Shigellosis: An invasive infection of the gastrointestinal tract, in Schessinger D, editor: Microbiology- 1975, Washington, DC, 1975, American Society for Microbiology. 8. Goldschmidt MC, and Du Pont HL: Enteropathogenic Escherichia coli: Lack of correlation of serotype with pathogenicity, J Infect Dis 133:153, 1976. 9. Ulshen MH, and Rollo JL: Pathogenesis of Escherichia coli gastroenteritis in man--another mechanism, N Engl J Med 302:99, 1980. 10. Cantey JR, and Blake RK: Diarrhea due to Escherichia coli in the rabbit: a novel mechanism, J Infect Dis 135:454, 1977. l l. Edwards PR, and Ewing WH: Identification of Enterobacteriaceae, Minneapolis, 1972, Burgess Publishing Company. 1Z Moseley SL, Hug I, Alim AR, S o M, Samadpour-Motalebi M, and Falkow S: Detection of enterotoxigenic Escherichia coli by DNA colony hybridization, J Infect Dis 142:892, 1980. 13. Polotsky YE, Dragunskaya EM, Seliverstova VG, et al: Pathogenic effect of cnterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea, Acta Microbiol Acta Sci Hung 24:221, 1977. 14. Levine MM, Berquist EJ, Nalin DR, Waterman DH, Hotnick RB, young CR, and Sotman S: Escherichia coli strains that cause diarrhea but do not produce heat-labile or heatstable enterotoxins and are non-invasive, Lancet 1:1119, 1978. 15. Klipstein FA, Rowe B, Engert RE, Short HB, and Gross R J: Enterotoxigenicity of enteropathogenic serotypes of Escherichia coli isolated from infants with epidemic diarrhea, Infect Immun 21:171, 1978. 16. Drucker MM, Polliach A, Yeivin R, and Sack TG: Immunofluorescent demonstration of enteropathogenic Escherichia coli in tissues of infants dying with enteritis, Pediatrics 46:855, I970. 17. Rothbaum R J, Partin JC, McAdams AJ, Shah DB, and Giannella RA: Enterocyte adherent Escherichia coli 0119:B 14: a novel mechanism of infant diarrhea, Gastroenterology 80:1265, 1981 (abstr). 18. Wachsmuth IK: Escherichia coil: Typing and pathogenicity, Clin Microbiol Newslett 2:4, 1980.
March 1982 TheJournalofPEDIATRICS
Chronic diarrhea in infants caused by adherent enteropathogenic Escherichia coli Enteropathogenic Escherichia coli (Ol l l:K58:H-) was isolated from the small bowel of two infants with chronic diarrhea. Small bowel biopsy revealed focal adherence of the bacteria to the epithelium, accompanied by inflammation of the tissue. The isolates also adhered to tissue culture cells in densely packed aggregates. The pattern o f adherence to tissue culture cells were found only among strains of serogroups 0111 and 0119 when 196 enteric E. coil isolates were tested. The pathogenic mechanism of enteropathogenic E. coil thus appears related to dense colonization of intestinal tissue. Adherent strains may be detected by tissue culture assay.
Carla R. Clausen, Ph.D.,* and Dennis L. Christie, M.D., Seattle, Wash.
ESCHERICHIA COL! m a y produce diarrhea by two welldefined mechanisms: Shigella-like penetration of intestinal epithelium, or elaboration of one or more enterotoxins. ~-4 T h e m e c h a n i s m responsible for infantile diarrhea associated with certain serotypes of E. coli (enteropathogenic E. coli) has not been established. A l t h o u g h some E P E C isolates have been found to produce enterotoxins or invade epithelial cells, 5-7 the majority of stains are neither toxigenic nor invasive. 8 A n o t h e r potential m e c h a n i s m was suggested by the isolation of an E. coli (0125 a t : H 2 1 ) from the small intestine of an infant with enterocolitis. 9 Electron microscopy of the duodenal biopsy specimen from the infant demonstrated bacilli adherent to the epithelium, with disruption of the microvilli. No enterotoxins nor invasive properties were associated with the isolate. The pathology of the infant's disease was similar to t h a t produced by a strain of E. coli which causes diarrhea in rabbits and adheres in large numbers to r a b b i t intestinal epithelium. ~~This strain did not produce any of the known enterotoxins and did not appear to invade the epithelial cells. In our hospital, E P E C 0111 was recently isolated from the small bowel of two infants with chronic diarrhea. T h e isolates adhered to tissue culture cells in densely packed From the Gastroenterology Division, Children's Orthopedic Hospital and Medical Center, *Reprint address: Department of Laboratories, COHMC, ~ P.O. Box C-5371, 4800 Sand Point Way N.E.; Seattle, WA 98105.
Vol. 100, No. 3, pp. 358-361
aggregates. Small bowel biopsy revealed acute inflammatory changes and focal adherence by large numbers of E P E C 0111. Abbreviations used EPEC: enteropathogenic Escherichia coli COHMC: Children's Orthopedic Hospital and Medical Center CASE REPORTS
Patients. Two infants from the ,same central Washington community were referred to Children's Orthopedic Hospital and Medical Center in Seattle for diarrhea which had been present for longer than two weeks. The first patient, 5.5 months of age, was admitted 8/12/80 following three weeks of persistent diarrhea and vomiting. The infant had been admitted to another hospital on two occasions because of dehydration. He continued to have 10 to 12 liquid stools per day and failed to tolerate clear liquids or soy or casein hydrolysate formulas. On admission to COHMC he was found to have a weight less than the third percentile, and a serum albumin concentration of 3 gm/dl. His daily stool output averaged 400 ml while fasting. He could not maintain his body weight and total parenteral nutrition was necessary. The diarrhea began to resolve 48 hours after intravenous gentamicin therapy was started. He was discharged to be fed a casein hydrolysate formula 30 days after admission; his weight gain was 1 kg. The second patient was 15 months of age when admitted 9/23/80, more than two weeks after the onset of diarrhea, vomiting, and low-grade fever. She had been admitted previously to another hospital because of dehydration. Prior to admission to COHMC, she had lost 1 kg in weight, Her serum albumin
0022-3476/82/030358+04500.40/0 9 1982 The C. V. Mosby Co.
Volume 100 Number 3
Diarrhea caused by adherent E. coli
359
concentration was 2.4 gm/dl. Stool output while fasting averaged 300 ml daily. She was given total parenteral nutrition to maintain body weight. The diarrhea began to resolve five days after initiation of intravenous gentamicin therapy. The patient was discharged 18 days after admission, receiving soy formula and a nonlactose-containing diet. Weight gain during hospitalization was 1.3 kg. Small bowel biopsy, performed on both patients the day after admission, was submitted for histologic examination. Intestinal fluid obtained at the same time was submitted for culture. METHODS Tissue culture assay. Leighton tubes containing No. 1 coverslips were seeded with cells of a continuous human epithelial cell line (Hep II). The cells were grown in Eagle minimal essential medium supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100 gg/ml streptomycin. When monolayers were nearly confluent, the medium was replaced with medium not containing antibiotics or serum. The monolayers were inoculated with 0.2 ml of an 18-hour bacterial culture grown in Trypticase soy broth. After a two-hour incubation at 35~ the medium was replaced with fresh medium. The cell culture was incubated an additional three to four hours. The coverslips were then removed from the tubes, washed vigorously in phosphatebuffered saline (pH 7.0), and stained by a Wright-Giemsa technique. Fluorescent antibody method. Sections of deparaffinated and acetone-fixed biopsy tissue were stained with dilutions of 0111:K58 sera prepared in rabbits (BBL Microbiology Systems, Cockeysville, Md.) Duplicate sections were treated with heterologous EPEC sera to provide negative controls. After incubation at 25~ for 30 minutes with the primary antisera, the sections were washed in phosphatebuffered saline, dipped into tap water, and blotted dry. All sections were then stained with a 1:200 dilution of fluorescein conjugated goat anti-rabbit globulin (Antibodies Incorporated, Davis, Calif.). After a 30-minute incubation, the sections were washed as before, dried, and mounted with buffered glycerol. RESULTS Culture isolates. Cultures of intestinal fluid from both infants yielded an abundant growth of non-motile E. coll. No anaerobes were isolated. Serotyping was performed by the methods described by Edwards and Ewing. ~ Heated suspensions of the isolates were agglutinated by a greater than 1:1,000 dilution of 0111:K58 antiserum. The 0111:K58:H- serotype was isolated from subsequent stool specimens from both infants. No other enteric pathogens, including Salmonella, Shigella, Yersinia enterocolitica, Campylobacter fetus subspecies jejuni, or rotavirus were detected. Polymorphonuclear leukocytes were observed in
Fig. 1. Section of small bowel biopsy stained with rabbit anti0111 sera and fluorescein-conjugatedanti-rabbit globulin. the stool from the second patient but not in stools from the first. Blood cultures were sterile. Neither isolate was found to contain genes which encode for heat labile or heat stable enterotoxin by DNA homology studies) z* Both were resistant to ampicillin, carbenicillin, cephalothin, kanamycin, sulfisoxazole, and neomycin, but susceptible to chloramphenieol, gentamicin, and tobramycin by disk diffusion assay. Cultures obtained when the biopsy was repeated two weeks later did not yield the EPEC. Small bowel biopsy. The small intestinal biopsies of the two patients showed a severe mucosal lesion. The lamina propria was edematous and densely populated with neutrophils, macrophages, and lymphocytes. No trophozoites of Giardia lamblia were found. Large numbers of bacilli were observed adhering to the epithelium, but no invasion into the lamina propria was evident. The bacteria were not found diffusely throughout the mucosa, but in discrete areas sometimes extending into the crypts. Fluorescent antibody staining identified the adherent bacilli as EPEC 0111 (Fig. 1). The repeat biopsy specimens showed *DNA homologystudieswere performedby S. k. Moseley,Department of Microbiologyand Immunology,Universityof Washington,Seattle, Washington.
360
Clausen and Christie
The Journal of Pediatrics March 1982 from the two infants in the present study plus an isolate from a third patient with chronic diarrhea. This patient (2.5 months of age) was referred to COHMC in June, 1980; EPEC 0 l l 1 was isolated from stool only and not from the small bowel. However, the biopsy was performed two weeks following a short course of ampicillin and gentamicin. Isolates from these three patients had the same multi-resistant antibiogram, and the three infants came to COHMC from the same community. DISCUSSION
Fig. 2. Aggregates of bacilli adhering to Hep II tissue culture cells. Wright-Giemsa stain.
improvement in villus architecture and an absence of bacteria. Tissue culture assay. The attachment of the EPEC 0111 isolates to tissue culture (Fig. 2) was not confluent but in aggregates, giving the appearance of forming microcolonies. Cytoplasmic invasion of cells, which can be demonstrated in the assay by virulent strains of Shigella, was not apparent. During the past several years, a number of enteric Strains of E. coli isolated from infants and children with diarrheal disease have been examined for adherence by the tissue culture assay. The isolates were selected from stool cultures that did not yield bacterial pathogens, i.e., Salmonella, Shigella, Y. enterocolitica, or C. fetus ss jejuni. Only 11 of 196 strains adhered to Hep II cells as described for the intestinal fluid isolates. Nonadherent strains were removed from the monolayer by the washing procedure, so that only an occasional bacterium was observed in the stained preparation. Six of the positive strains were serotype 011 l:K58:H-. The other five were motile strains which agglutinated in 0119:K69 antiserum. The H (flagellar) antigen was n(~t determined. The six positive 0111 isolates include those
Enteropathogenic E. coli is not a homogeneous group of pathogens, since some strains of 026:H 11 J3 and 0128:H126 have been shown to elaborate enterotoxins, and strains of serogroup 028 are capable of Shigella-like invasion of cells, 7 The pathogenic mechanism for the majority of EPEC remains obscure. For example, two strains (0142:H6 and 0127:H6) isolated during outbreaks of infantile diarrhea produced a diarrheal illness in adult volunteers. Tests for heat-labile and heat-stable enterotoxin, as well as for invasiveness, were negative?4 However, there is some indication that these two isolates may induce fluid secretion from the bowel by another as yet uncharacterized toxin? 5 The present study reinforces evidence that infection with certain strains of EPEC may result in serious diarrhea; the pathogenesis appears related to tissue adherence. Adherence by strains of serogroup 0111 has been noted in several studies. By immunofluorescence, Drucker et a116 found EPEC attached to intestinal tissue in sections obtained at autopsy of 17 infants who had died of enteritis; 14 of the 17 were serogroup 0111. All of the EPEC-associated fatal cases had inflammatory changes in the intestine ranging from moderate (chronic inflammation with flattened villi) to severe (acute inflammation and mucosal necrosis). Polotsky et al ~3 illustrated by electron microscopy the attachment of some strains of EPEC, including 0111, to rabbit intestine. Rothbaum et a117 recently described an outbreak of infantile diarrhea caused by 0119:B 14(K69); the organism was isolated from both stool and duodenal aspirates. Bacteria were seen adhering in clusters to the mucosal surfaces of atrophic villi in small bowel biopsy tissue. Some strains of 0111 and 0119 possess virulence factors which enable them to colonize intestinal epithelial cells. Although a heretofore unrecognized toxin may mediate the subsequent diarrheal illness, the pathology noted in our patients does not correlate with the toxigenic disease, which leaves the bowel intact. Disruption of villi and inflammatory changes may be caused solely by the effect of firm attachment of the organism, with subsequent exposure of the host tissue to enzymatic activity and
Volume 1O0 Number 3
metabolic products of the a d h e r e n t bacteria. Dense colonization could conceivably interfere with normal absorption and with secretory processes of the intestinal epithelium. Finally, the a d h e r e n t strains can be detected in tissue culture assay. T h e p a t t e r n of adherence is unique and was found in only 11 of 196 enteric isolates of E. coli. All of the a d h e r e n t E. coli were in serogroups 0111 and 0119. T h e six 0111 isolates which adhered to Hep II cells were nonmotile, thus representing a serotype commonly associated with infantile d i a r r h e a (0111 :K58:H-). T h e a d h e r e n t 0119 strains were not completely characterized since ttagellar antigen typing was not performed. Thus it is not known if these isolates were 0119:K69:H6, which is the only serotype of the serogroup 0119 recognized as an E P E C . TM T h e assay reflected in vivo adherence to intestinal t i s s u e o f the two infants in the present study, and m a y serve as a tool to detect virulent strains of enteric E. coll. REFERENCES 1. Du Pont HL, Formal SB, Hornick RB, Snyder M J, Libonati JP, Sheahan DG, Le Brec EH, and Kalas JP: Pathogenesis of Escherichia coli diarrhea, N Engl J Med 285:1, 1971. 2. Sack RB, Gorbach SL, Banwell JG, Jacobs B, Chatterjee BD, and Mitra RC: Enterotoxigenic Escherichia coil isolated from patients with severe cholera-like disease. J Infect Dis 123:378, 1971. 3. Gorbach SL, Kean BH, Evans DG, Evans D J, and Bessudo D: Travelers' diarrhea and toxigenic Escherichia coli, N Engl J Med 292:933, 1975. 4. Sack DA, Merson MH, Wells JG, Sack RB, and Morris GK: Diarrhoea associated with heat-stable enterotoxin-producing strains of Escherichia coli, Lancet 2:239, 1975. 5. Gorbach SL, and Khurana CM: Toxigenic Escherichia coil: A cause of infantile diarrhea in Chicago, N Engl J Med 287:79t, 1972. 6. Reis MH, Castro AF, Toledo RM, and Trabulsi LR: Production of heat-stable enterotoxin by the 0128 serogroup of Escherichia coli, Infect Immun 24:289, 1979.
Diarrhea caused by adherent E. coli
36 1
7. Gemski P Jr, and Formal SB: Shigellosis: An invasive infection of the gastrointestinal tract, in Schessinger D, editor: Microbiology- 1975, Washington, DC, 1975, American Society for Microbiology. 8. Goldschmidt MC, and Du Pont HL: Enteropathogenic Escherichia coli: Lack of correlation of serotype with pathogenicity, J Infect Dis 133:153, 1976. 9. Ulshen MH, and Rollo JL: Pathogenesis of Escherichia coli gastroenteritis in man--another mechanism, N Engl J Med 302:99, 1980. 10. Cantey JR, and Blake RK: Diarrhea due to Escherichia coli in the rabbit: a novel mechanism, J Infect Dis 135:454, 1977. l l. Edwards PR, and Ewing WH: Identification of Enterobacteriaceae, Minneapolis, 1972, Burgess Publishing Company. 1Z Moseley SL, Hug I, Alim AR, S o M, Samadpour-Motalebi M, and Falkow S: Detection of enterotoxigenic Escherichia coli by DNA colony hybridization, J Infect Dis 142:892, 1980. 13. Polotsky YE, Dragunskaya EM, Seliverstova VG, et al: Pathogenic effect of cnterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea, Acta Microbiol Acta Sci Hung 24:221, 1977. 14. Levine MM, Berquist EJ, Nalin DR, Waterman DH, Hotnick RB, young CR, and Sotman S: Escherichia coli strains that cause diarrhea but do not produce heat-labile or heatstable enterotoxins and are non-invasive, Lancet 1:1119, 1978. 15. Klipstein FA, Rowe B, Engert RE, Short HB, and Gross R J: Enterotoxigenicity of enteropathogenic serotypes of Escherichia coli isolated from infants with epidemic diarrhea, Infect Immun 21:171, 1978. 16. Drucker MM, Polliach A, Yeivin R, and Sack TG: Immunofluorescent demonstration of enteropathogenic Escherichia coli in tissues of infants dying with enteritis, Pediatrics 46:855, I970. 17. Rothbaum R J, Partin JC, McAdams AJ, Shah DB, and Giannella RA: Enterocyte adherent Escherichia coli 0119:B 14: a novel mechanism of infant diarrhea, Gastroenterology 80:1265, 1981 (abstr). 18. Wachsmuth IK: Escherichia coil: Typing and pathogenicity, Clin Microbiol Newslett 2:4, 1980.