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Culture supernatant of Shiga toxin-producing Escherichia coli strains provoke fluid accumulation in rabbit ileal loops
FEMS Immunology and Medical Microbiology 19 (1998) 285^288
Culture supernatant of Shiga toxin-producing Escherichia coli strains provoke £uid accumulation in rabbit ileal loops Antonio J.P. Ferreira a , Waldir P. Elias Jr. b , Jacinta S. Pelayo c , Regina Giraldi b , Margareth Z. Pedroso b , Isabel C.A. Scaletsky b; * a
Departamento de Patologia, Faculdade de Medicina Veterinaèria e Zootecnia, Universidade de Saìo Paulo, Saìo Paulo, Brazil b Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Saìo Paulo, Rua Botucatu 862, 04023-062 Saìo Paulo, SP, Brazil c Departamento de Patologia Geral, Universidade Estadual de Londrina, Londrina, Brazil Received 15 July 1997; revised 20 October 1997; accepted 20 October 1997
Abstract Production of Shiga toxin (Stx) in Escherichia coli strains belonging to serogroups O26, O111, and O157 was evaluated in the rabbit ileal loop assay and results were compared to those using tissue culture assays and DNA hybridization with specific probes for Stx1 and Stx2. All 14 Shiga toxin-producing E. coli strains tested provoked fluid accumulation in the rabbit intestinal loop. Eleven strains hybridized with Stx1 probe, one strain with Stx2 and two strains with both probes. Filtered culture supernatants of all E. coli strains presented cytotoxic effects in both HeLa and Vero cells. In this study, we found a strong association between the production of Stx and its effect in an animal model. This is the first description of high-level Stxproducing E. coli O111ac isolated in Brazil. z 1998 Published by Elsevier Science B.V. All rights reserved. Keywords : Escherichia coli; Shiga-like toxin; Rabbit ileal loop assay
1. Introduction Verotoxins (VTs) or Shiga-like toxins (SLTs) are closely related A-B subunit toxins produced by certain strains of Escherichia coli. Both terms have been used synonymously and were recently designated as Shiga toxin (Stx) [1]. Two principal groups of these toxins have been described, Stx1 (VT-1/SLT-I) and Stx2 (VT-2/SLT-II); both toxins are encoded by bacteriophages. All of these toxins present similar bio* Corresponding author. Tel.: +55 (11) 50843213; Fax: +55 (11) 5716504; E-mail: [email protected]/[email protected]
logical e¡ects but di¡er signi¢cantly in immunological reactivity [2,3]. Normally, the cytotoxic e¡ects of these toxins are detected by using tissue culture assays, employing Vero or HeLa cells [4,5]. In epidemiological studies, DNA hybridization with speci¢c probes is commonly used to detect Stx positive strains [6]. More recently, polymerase chain reaction (PCR) ampli¢cation has been used for detection of E. coli Stx genes in bacterial strains present in stool specimens, without prior culture [7]. E. coli strains producing Stx1 and Stx2 have been closely associated with food-borne outbreaks of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Although, the ¢rst description of
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Stx production was in E. coli serotype O26:H11 [4], cytotoxin-producing E. coli have been detected mainly in strains of the serotype O157:H7, the principal member of the enterohemorrhagic E. coli (EHEC) group [3]. Moreover, E. coli strains of other serogroups have also been associated to these cytotoxins. Several of these strains are considered classical enteropathogenic E. coli (EPEC) serogroups, such as O55, O111 and O119 [8]. The purpose of this study was to evaluate the response of the rabbit ileal loop to cytotoxin-producing E. coli. The rabbit ileal loop assay described by De and Chatterje [9] has been used to indicate enterotoxin activity in culture supernatants of enterotoxin-producing enteropathogens such as E. coli enterotoxigenic (ETEC), Vibrio cholerae [9,10] and of puri¢ed toxin of EHEC [11].
2. Material and methods 2.1. Strains We selected for this study 14 E. coli strains which were previously identi¢ed as Stx producing [12,13]. Four strains belong to serotype O26:H11, 4 to serotype O157:H7 and 6 to serogroup O111ac:H8. Four of the 6 O111ac strains were isolated in Brazil from cases of infantile diarrhea [12]. All of the other strains were isolated in di¡erent countries and were kindly provided by Dr. Thomas S. Whittam (Department of Biology, The Pennsylvania State University) Table 1 Sources and £agellar antigens (H types) of E. coli strains studied Serotype
No. of strains
Source
Origin/Period
O26:H11
1 1 1 1 3 2 1 2 1 1
Human Human Human/IDb Calf Human/ID Human/ID Human/ID Human/ID Human/ID Calf
Canada/NDa Australia/1986 USA/1977 USA/1990 Brazil/1987,1989,1989 USA/1964,1977 Brazil/1993 USA/1988,1990 Denmark/1988 Argentina/1977
O111ac :H3 O111ac :H8 O157:H7
a b
Not determined. Infantile diarrhea.
[13] (Table 1). All strains were tested for the presence of Stx genes and for cytotoxin production in tissue culture assays prior to performing the rabbit ileal loop assay. Bacterial strains were grown aerobically at 37³C for 24 h in Trypticase Soy Broth (TSB, Difco) and Evans medium [10] for tissue culture and rabbit ileal loop assays, respectively. The cultures were centrifuged (8000Ug for 20 min) and ¢ltered through 0.22 Wm Millipore membrane. 2.2. Cytotoxin production Cytotoxin production was detected by the tissue culture assay described previously by Konowalchuk et al. [4]. HeLa and Vero cell monolayers were obtained by seeding 105 cells in 96 well tissue culture plates. After 24 h of incubation at 37³C in 5% CO2 atmosphere, ¢ltered culture supernatants were diluted 1:4 and added to the cell monolayers. Plates were maintained at the conditions described above for 24 to 72 h to detect cytotoxic e¡ect. 2.3. DNA hybridization Strains were tested for the presence of Stx genes by colony blot hybridization [14]. The Stx1 probe was a 1142 bp BamHI fragment from plasmid pJN37-19, and Stx2 probe was an 842 bp SmaI-PstI fragment of pNN111-19 [15]. The DNA probe fragments were labelled with [K-32 P]dATP by nick translation [16]. 2.4. Rabbit ileal loop assay The rabbit ileal loop assay was performed in duplicate in 9 week-old white rabbits according to the method of Moon et al. [17]. Brie£y, 1 ml of ¢ltered culture supernatant was injected into each 5 cm ileal loop and £uid accumulation was observed after 18 h. In the autopsy, the macroscopic aspect of the loops was recorded. The presence of gross lesion and volume of £uid in the ileal loops was graded as +, mild; ++, moderate; +++, marked; ++++, severe. E. coli strain 40T, kindly provided by Dr. Beatriz E.C. Guth (Disciplina de Microbiologia, Universidade Federal de Saìo Paulo) was used as an elevated level-producing control and the Evans medium as negative control.
FEMSIM 819 6-1-98
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Table 2 Detection of cytotoxic activity in E. coli strains by tissue culture, DNA hybridization and rabbit ileal loop Serotype O111ac :H3 O111ac :H8 O26:H11 O157:H7 O157:H7 O157:H7 Positive controlb Negative controlc
No. of strains
5 1 4 2 1 1
Tissue culturea
+ + + + + + + 3
DNA hybridization Stx1
Stx2
+ + + + + 3 + 3
3 3 3 + 3 + + 3
Rabbit ileal loop
+ + + + + + + 3
a
Cytotoxic e¡ect detected by HeLa and Vero cells. E. coli 40T. c Evans medium. b
3. Results The results of the DNA hybridization tests, tissue culture and rabbit ileal loop assays are summarized in Table 2. All strains provoked £uid accumulation in the rabbit intestinal loop, indicating production of Stx1, Stx2 or both. 3.1. HeLa and Vero cell assays Filtered culture supernatants of 14 E. coli strains produced cytotoxic e¡ects in both HeLa and Vero cells. The cytotoxic doses required to kill 50% of the cells (CD 50/ml) varied from 103 to 104 . The ¢ltered culture supernatant e¡ects of the O111 strains were neutralized by anti-Stx1 antiserum (data not shown). Of these cytotoxin-producing strains, 11 hybridized with Stx1 probe, 1 strain hybridized with Stx2 probe and 2 strains reacted with both probes (Table 2). 3.2. Rabbit ileal loop assay All 14 cytotoxin-producing E. coli strains caused £uid accumulation with evident dilation of the ileal loops. These results were consistent in at least two repetitive tests. When compared with controls these positive strains presented a discrete variation of the intestinal dilation intensity (+++ to ++++).
4. Discussion
time that the detection of Stxs in culture supernatant ¢ltrates can be demonstrated in the rabbit ileal loop assay. This assay indicates £uid accumulation in ligated intestinal segments due to the presence of an enteric toxin secreted in bacterial cultures [9,10]. A good accordance was observed between the results obtained in the three di¡erent assays employed. This was also observed in previous studies that used tissue culture and hybridization probe assays [8,18]. In the past, some authors showed £uid accumulation in rabbit ileal loops inoculated with culture or supernatant of EPEC strains of di¡erent O serogroups [19^21]. It is now known that typical EPEC do not produce Stx. It is probable that these authors were working with Stx-producing strains belonging to these serogroups. Although the strains analyzed were from di¡erent parts of the world, it is very interesting to note that 4 cytotoxin-producing strains were isolated in Brazil. In previous studies, performed in our country, the majority of the E. coli strains analyzed were noncytotoxin producing [22]. A low cytotoxic activity, which could be neutralized with anti-Stx1 antisera, was found in sonic lysates of E. coli O111:H3 , O111:H2 and O119:H6 serotypes [23]. Another EPEC O111ab:H3 strain was found to be Stx2 probe positive but no cytotoxic activity was detected in the culture ¢ltrates [24]. This is the ¢rst description of high-level cytotoxin production in the serogroup O111 isolated from children with diarrhea in our community.
In the present study we have shown for the ¢rst
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Acknowledgments This study was supported by Fundac°aìo de Amparo aé Pesquisa do Estado de Saìo Paulo (FAPESP), and by Conselho Nacional de Desenvolvimento Cient|è¢co e Tecnoèlogico (CNPq).
References
[13]
[14]
[15]
[1] Calderwood, S.B., Acheson, D.W.K., Keusch, G.T., Barrett, T.J., Gri¤n, P.M., Strockbine, N.A., Swaminathan, B., Kaper, J.B., Levine, M.M., Kaplan, B.S., Karch, H., O'Brien, A.D., Obrig, T.G., Takeda, Y., Tarr, P.I. and Wachsmuth, I.K. (1996) Proposed new nomenclature for SLT (VT) family. ASM News 62, 118^119. [2] Gyles, C.L. (1992) Escherichia coli cytotoxins and enterotoxins. Can. J. Microbiol. 38, 734^746. [3] Karmali, M.A. (1989) Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2, 15^38. [4] Konowalchuk, J., Speirs, J.I. and Stavric, S. (1977) Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18, 775^779. [5] O'Brien, A.D., LaVeck, G.D., Thompson, M.R. and Formal, S.B. (1982) Characterization of Shigella dysenteriae 1 (Shiga) toxin puri¢ed by anti-Shiga toxin a¤nity chromatography. J. Infect. Dis. 146, 763^769. [6] Gomes, T.A.T., Rassi, V., MacDonald, K.L., Ramos, S.R.T.S., Trabulsi, L.R., Vieira, M.A.M., Guth, B.E.C., Candeias, J.A.N., Ivey, C., Toledo, M.R.F. and Blake, P.A. (1991) Enteropathogens associated with acute diarrheal disease in urban infants in Saìo Paulo, Brazil. J. Infect. Dis. 164, 331^ 337. [7] Brian, M.J., Frosolono, M., Murray, B.E., Miranda, A., Lopez, E.L., Gomez, H.F. and Cleary, T.G. (1992) Polymerase chain reaction for diagnosis of enterohemorragic Escherichia coli infection and hemolytic-uremic syndrome. J. Clin. Microbiol. 30, 1801^1806. [8] Bitzan, M., Karch, H., Maas, M.G., Meyer, T., Russmann, H., Aleksic, S. and Bockemuhl, J. (1991) Clinical and genetic aspects of Shiga-like toxin production in traditional enteropathogenic Escherichia coli. Zent.bl. Bakt. 274, 496^506. [9] De, S.N. and Chatterje, D.N. (1953) An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J. Pathol. Bact. 66, 559^562. [10] Evans, D.G., Evans Jr., D.J. and Pierce, N.F. (1973) Di¡erences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli. Infect. Immun. 7, 873^880. [11] Keenan, K.P., Sharpnack, D.D., Collins, H., Formal, S.B. and O'Brien, A.D. (1986) Morphologic evaluation of the effects of Shiga toxin and E. coli Shiga-like toxin on the rabbit intestine. Am. J. Pathol. 125, 69^80. [12] Campos, L.C., Whittam, T.S., Gomes, T.A.T., Andrade,
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
J.R.C. and Trabulsi, L.R. (1994) Escherichia coli serogroup O111 includes several clones of diarrheagenic strains with different virulence properties. Infect. Immun. 62, 3282^3288. Whittam, T.S., Wolfe, M.L., Wachsmuth, I.K., Orskov, F., Orskov, I. and Wilson, R.A. (1993) Clonal relationships among Escherichia coli strains that cause hemorragic colitis and infantile diarrhea. Infect. Immun. 61, 1619^1629. Maas, R. (1983) An improved colony hybridization method with signi¢cantly increased sensitivity for detection of single genes. Plasmid 10, 296^298. Newland, J.W. and Neill, R.J. (1988) DNA probes for Shigalike toxins I and II and for toxin-converting bacteriophages. J. Clin. Microbiol. 26, 1292^1297. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Moon, H.W., Whipp, S.C., Argenzio, R.A., Levine, M.M. and Gianella, R.A. (1983) Attaching and e¡acing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun. 41, 1340^1351. Beutin, L., Montenegro, M.A., Orskov, I., Orskov, F., Prada, J., Zimmermann, S. and Stephan, R. (1989) Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J. Clin. Microbiol. 27, 2559^2564. Polotsky, Y.E., Dragunskaya, E.M., Seliverstova, V.G., Avdeeva, T.A., Chakhutinskaya, M.G., Keèty, I., Vertenyi, A., Ralovich, B., Emody, L., Malovics, I., Satonova, N.V., Snigirevskaya, E.S. and Karyagina, E.I. (1977) Pathogenic e¡ect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea. Acta Microbiol. Acad. Sci. Hung. 24, 221^ 236. Trabulsi, L.R. (1964) Revelac,aìo de colibacilos associados aés diarreèias infantis pelo meètodo da infecc,aìo experimental da alc,a ligada de intestino de coelho. Rev. Inst. Med. Trop. Sao Paulo 6, 197^203. Zinkernagel, R.M. and Colombini, A. (1975) Passive oral immunization with immunoglobulins: enteropathogenic Escherichia coli from infants and bovine anti-E. coli lactoserum assayed in the rabbit ileal loop model. Med. Microbiol. Immunol. 162, 1^7. Silva, M.L.M., Scaletsky, I.C.A. and Viotto, L.H. (1983) Non producing of cytotoxin among enteropathogenic strains of Escherichia coli isolated in Saìo Paulo, Brasil. Rev. Microbiol. 14, 161^162. Giraldi, R., Guth, B.E.C. and Trabulsi, L.R. (1990) Production of Shiga-like toxin among Escherichia coli strains and other bacteria isolated from diarrhea in Saìo Paulo, Brazil. J. Clin. Microbiol. 28, 1460^1462. Guth, B.E.C., Giraldi, R., Gomes, T.A.T. and Marques, L.R.M. (1994) Survey of cytotoxin production among Escherichia coli strains characterized as enteropathogenic (EPEC) by serotyping and presence of EPEC adherence factor (EAF) sequences. Can. J. Microbiol. 40, 341^344.
FEMSIM 819 6-1-98
Culture supernatant of Shiga toxin-producing Escherichia coli strains provoke £uid accumulation in rabbit ileal loops Antonio J.P. Ferreira a , Waldir P. Elias Jr. b , Jacinta S. Pelayo c , Regina Giraldi b , Margareth Z. Pedroso b , Isabel C.A. Scaletsky b; * a
Departamento de Patologia, Faculdade de Medicina Veterinaèria e Zootecnia, Universidade de Saìo Paulo, Saìo Paulo, Brazil b Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Saìo Paulo, Rua Botucatu 862, 04023-062 Saìo Paulo, SP, Brazil c Departamento de Patologia Geral, Universidade Estadual de Londrina, Londrina, Brazil Received 15 July 1997; revised 20 October 1997; accepted 20 October 1997
Abstract Production of Shiga toxin (Stx) in Escherichia coli strains belonging to serogroups O26, O111, and O157 was evaluated in the rabbit ileal loop assay and results were compared to those using tissue culture assays and DNA hybridization with specific probes for Stx1 and Stx2. All 14 Shiga toxin-producing E. coli strains tested provoked fluid accumulation in the rabbit intestinal loop. Eleven strains hybridized with Stx1 probe, one strain with Stx2 and two strains with both probes. Filtered culture supernatants of all E. coli strains presented cytotoxic effects in both HeLa and Vero cells. In this study, we found a strong association between the production of Stx and its effect in an animal model. This is the first description of high-level Stxproducing E. coli O111ac isolated in Brazil. z 1998 Published by Elsevier Science B.V. All rights reserved. Keywords : Escherichia coli; Shiga-like toxin; Rabbit ileal loop assay
1. Introduction Verotoxins (VTs) or Shiga-like toxins (SLTs) are closely related A-B subunit toxins produced by certain strains of Escherichia coli. Both terms have been used synonymously and were recently designated as Shiga toxin (Stx) [1]. Two principal groups of these toxins have been described, Stx1 (VT-1/SLT-I) and Stx2 (VT-2/SLT-II); both toxins are encoded by bacteriophages. All of these toxins present similar bio* Corresponding author. Tel.: +55 (11) 50843213; Fax: +55 (11) 5716504; E-mail: [email protected]/[email protected]
logical e¡ects but di¡er signi¢cantly in immunological reactivity [2,3]. Normally, the cytotoxic e¡ects of these toxins are detected by using tissue culture assays, employing Vero or HeLa cells [4,5]. In epidemiological studies, DNA hybridization with speci¢c probes is commonly used to detect Stx positive strains [6]. More recently, polymerase chain reaction (PCR) ampli¢cation has been used for detection of E. coli Stx genes in bacterial strains present in stool specimens, without prior culture [7]. E. coli strains producing Stx1 and Stx2 have been closely associated with food-borne outbreaks of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Although, the ¢rst description of
0928-8244 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved. PII S 0 9 2 8 - 8 2 4 4 ( 9 7 ) 0 0 0 9 4 - 1
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Stx production was in E. coli serotype O26:H11 [4], cytotoxin-producing E. coli have been detected mainly in strains of the serotype O157:H7, the principal member of the enterohemorrhagic E. coli (EHEC) group [3]. Moreover, E. coli strains of other serogroups have also been associated to these cytotoxins. Several of these strains are considered classical enteropathogenic E. coli (EPEC) serogroups, such as O55, O111 and O119 [8]. The purpose of this study was to evaluate the response of the rabbit ileal loop to cytotoxin-producing E. coli. The rabbit ileal loop assay described by De and Chatterje [9] has been used to indicate enterotoxin activity in culture supernatants of enterotoxin-producing enteropathogens such as E. coli enterotoxigenic (ETEC), Vibrio cholerae [9,10] and of puri¢ed toxin of EHEC [11].
2. Material and methods 2.1. Strains We selected for this study 14 E. coli strains which were previously identi¢ed as Stx producing [12,13]. Four strains belong to serotype O26:H11, 4 to serotype O157:H7 and 6 to serogroup O111ac:H8. Four of the 6 O111ac strains were isolated in Brazil from cases of infantile diarrhea [12]. All of the other strains were isolated in di¡erent countries and were kindly provided by Dr. Thomas S. Whittam (Department of Biology, The Pennsylvania State University) Table 1 Sources and £agellar antigens (H types) of E. coli strains studied Serotype
No. of strains
Source
Origin/Period
O26:H11
1 1 1 1 3 2 1 2 1 1
Human Human Human/IDb Calf Human/ID Human/ID Human/ID Human/ID Human/ID Calf
Canada/NDa Australia/1986 USA/1977 USA/1990 Brazil/1987,1989,1989 USA/1964,1977 Brazil/1993 USA/1988,1990 Denmark/1988 Argentina/1977
O111ac :H3 O111ac :H8 O157:H7
a b
Not determined. Infantile diarrhea.
[13] (Table 1). All strains were tested for the presence of Stx genes and for cytotoxin production in tissue culture assays prior to performing the rabbit ileal loop assay. Bacterial strains were grown aerobically at 37³C for 24 h in Trypticase Soy Broth (TSB, Difco) and Evans medium [10] for tissue culture and rabbit ileal loop assays, respectively. The cultures were centrifuged (8000Ug for 20 min) and ¢ltered through 0.22 Wm Millipore membrane. 2.2. Cytotoxin production Cytotoxin production was detected by the tissue culture assay described previously by Konowalchuk et al. [4]. HeLa and Vero cell monolayers were obtained by seeding 105 cells in 96 well tissue culture plates. After 24 h of incubation at 37³C in 5% CO2 atmosphere, ¢ltered culture supernatants were diluted 1:4 and added to the cell monolayers. Plates were maintained at the conditions described above for 24 to 72 h to detect cytotoxic e¡ect. 2.3. DNA hybridization Strains were tested for the presence of Stx genes by colony blot hybridization [14]. The Stx1 probe was a 1142 bp BamHI fragment from plasmid pJN37-19, and Stx2 probe was an 842 bp SmaI-PstI fragment of pNN111-19 [15]. The DNA probe fragments were labelled with [K-32 P]dATP by nick translation [16]. 2.4. Rabbit ileal loop assay The rabbit ileal loop assay was performed in duplicate in 9 week-old white rabbits according to the method of Moon et al. [17]. Brie£y, 1 ml of ¢ltered culture supernatant was injected into each 5 cm ileal loop and £uid accumulation was observed after 18 h. In the autopsy, the macroscopic aspect of the loops was recorded. The presence of gross lesion and volume of £uid in the ileal loops was graded as +, mild; ++, moderate; +++, marked; ++++, severe. E. coli strain 40T, kindly provided by Dr. Beatriz E.C. Guth (Disciplina de Microbiologia, Universidade Federal de Saìo Paulo) was used as an elevated level-producing control and the Evans medium as negative control.
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Table 2 Detection of cytotoxic activity in E. coli strains by tissue culture, DNA hybridization and rabbit ileal loop Serotype O111ac :H3 O111ac :H8 O26:H11 O157:H7 O157:H7 O157:H7 Positive controlb Negative controlc
No. of strains
5 1 4 2 1 1
Tissue culturea
+ + + + + + + 3
DNA hybridization Stx1
Stx2
+ + + + + 3 + 3
3 3 3 + 3 + + 3
Rabbit ileal loop
+ + + + + + + 3
a
Cytotoxic e¡ect detected by HeLa and Vero cells. E. coli 40T. c Evans medium. b
3. Results The results of the DNA hybridization tests, tissue culture and rabbit ileal loop assays are summarized in Table 2. All strains provoked £uid accumulation in the rabbit intestinal loop, indicating production of Stx1, Stx2 or both. 3.1. HeLa and Vero cell assays Filtered culture supernatants of 14 E. coli strains produced cytotoxic e¡ects in both HeLa and Vero cells. The cytotoxic doses required to kill 50% of the cells (CD 50/ml) varied from 103 to 104 . The ¢ltered culture supernatant e¡ects of the O111 strains were neutralized by anti-Stx1 antiserum (data not shown). Of these cytotoxin-producing strains, 11 hybridized with Stx1 probe, 1 strain hybridized with Stx2 probe and 2 strains reacted with both probes (Table 2). 3.2. Rabbit ileal loop assay All 14 cytotoxin-producing E. coli strains caused £uid accumulation with evident dilation of the ileal loops. These results were consistent in at least two repetitive tests. When compared with controls these positive strains presented a discrete variation of the intestinal dilation intensity (+++ to ++++).
4. Discussion
time that the detection of Stxs in culture supernatant ¢ltrates can be demonstrated in the rabbit ileal loop assay. This assay indicates £uid accumulation in ligated intestinal segments due to the presence of an enteric toxin secreted in bacterial cultures [9,10]. A good accordance was observed between the results obtained in the three di¡erent assays employed. This was also observed in previous studies that used tissue culture and hybridization probe assays [8,18]. In the past, some authors showed £uid accumulation in rabbit ileal loops inoculated with culture or supernatant of EPEC strains of di¡erent O serogroups [19^21]. It is now known that typical EPEC do not produce Stx. It is probable that these authors were working with Stx-producing strains belonging to these serogroups. Although the strains analyzed were from di¡erent parts of the world, it is very interesting to note that 4 cytotoxin-producing strains were isolated in Brazil. In previous studies, performed in our country, the majority of the E. coli strains analyzed were noncytotoxin producing [22]. A low cytotoxic activity, which could be neutralized with anti-Stx1 antisera, was found in sonic lysates of E. coli O111:H3 , O111:H2 and O119:H6 serotypes [23]. Another EPEC O111ab:H3 strain was found to be Stx2 probe positive but no cytotoxic activity was detected in the culture ¢ltrates [24]. This is the ¢rst description of high-level cytotoxin production in the serogroup O111 isolated from children with diarrhea in our community.
In the present study we have shown for the ¢rst
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Acknowledgments This study was supported by Fundac°aìo de Amparo aé Pesquisa do Estado de Saìo Paulo (FAPESP), and by Conselho Nacional de Desenvolvimento Cient|è¢co e Tecnoèlogico (CNPq).
References
[13]
[14]
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