Castro, A. F. Pestana de

Pustiglione Neto, L.

Rangel, H. A.

Serafim, M. B.

English

Cadernos Espinosanos (E-Journal)

Rev. lnst. Med. trop. São Paulo 19(3): 144-147, maio-junho, 1977 UDC 616.981 616.34-002 ISOLATION OF HEAT-STABLE ENTEROTOXIN-PRODUCING STRAINS OF ESCHERICHIA COLI ISOLATED FROM CASES OF DIARRHEA IN CAMPINAS, SP, BRAZIL lU. B. SERAFIM (1), A. F. PESTANA DE CASTRO (1), H. A. RANGEL (1) and L. PUSTIGLIONE NETO (2) SUMMARY The Authors report the occurrence of three strains of Escherichia coli producing only toxin of the ST type, revealed by DEAN'S test. When tested in Yl cells and in rab-bit skin, theses strains showed no production of LT toxin. ln all cases the ST-producing strains provided an intestine to body weight ratios equal or greater than 0.122. On the other hand, 21 ST-negative strains produced ratios between O. 050 and O. 059 followed by 15 strains which gave values between 0.060 and O. 069. Onl'.Y two negative strains evoked reactions between O. 070 and O. 075. INTRODUCTION Diarrheal disease is one of the leading causes of death among children under three years of age, who live in developing coun-tries 7 • Bacteriological examinations of diarrheal stools in different countries have often given similar results, characterized by the absence, in many cases, of the known recognized ente-ropathogens. Since 1956, after DE et al. 1 it has become clear that some colibacilli, not necessarily be-longing to the classic enteropathogenic se-rotypes, were able to cause dilatation and in-creased volumes of fluid in the ligated intes-tinal ileal loop test performed in adult rabb-its. Later, other Authors s,s,9, besides confirm-ing these findings, found that this picture could be reproduced With bacteria-free filtra-tes of known E. coli strains which had been isolated from pigs and calves with diarrhea and had been found to produce dilatation when injected into the ligated intestine as live bacterial cultures. Based upon these ex-periments it was conclude<[ that at least in those cases enteropathogenicity was due to the production of an enterotoxin. Human and swine enteropathogenic Es-cherichia coli are now known to produce at least two types of enterotoxin. One, heat la-bile (LT), similar, if not identical, to cholera toxin, with high molecular weight, is able to give positive results in the adult rabbit ileal loop 7 • As it occurs with cholera toxin, su-pernatants either concentrated or not of the-se E. coli strains when inoculated intrader-micaliy in the rabbit depilated skin caused lo-calized induration and increased permeabili-ty of small blood vessels. This property of L T toxin of E. coli, named permeability factor (PF) and first observed with cholera toxin, may be used for its characterization and mea-surement 6. Recently it has been demonstrated that bacteria-free filtrates of these microorga-Supported by the «Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Grant 2222-0732/75. (1) Department of Microbiology and Immunology, University of Campinas, SP, Brasil. (2) Biological Institute, São Paulo, SP, Brasil. 144 SERAFIM, M. B.; PESTANA DE CASTRO, A. F.; RANGEL, H. A. & PUSTIGLIONE NETO, L. - Isolation of heat-stable enterotoxin-producing strains of Escherichia- coli isolated from cases of diarrhea in Campinas, SP, Brazil. Rev. Inst. Med. tI·op. São Paulo 19:144-147, 1977. nisms were able to cause rounding of the elon-gated Yl adrenal cells 3 • This effect was not anymore observed when these preparations were previously heated at 100°0 for 15 minu-tes. On account cit several experiments carri-ed out first with E. coli strains isolated from animals and later confirmed for human strains, it was verified that colibacilli may produce a low-molecular weight heat-stable toxin which can also cause, though more ra-pidly, a positive rabbit ileal loop test 9. ST toxin as reported by DEAN et a1.2 is also able to evoke the sarne picture in the intestines of infant mice. Qpposite to LT toxin, ST pro-duces neither rounding ofYl cells nor PF 1. With exception to Trabulsi's paper (1964 10, • São Paulo, Brazil) in which the occurrence of enterotoxigenic E. coli has been demonstrat-ed and rei,earches 11 presently being éa:r;ried out at the Department of Microbiolog_y and Parasitology of the "Escola Paulista de Medi-cina", SP, Brazil, nothing was found regard-ing other reports on the existence of ST to-xin-prod ucing strains of E. coli in Brazil, de-tected by DEAN's technique. Due to those considerations and to the importance of the subject we think it is op-portune to report the occurrence in Campinas, SP, Brazil, of diarrhea in children under three years old, whose stools revealed the presence of ST enterotoxin-producing strains of Esche-richia coli at the bacteriological examinations. MATERIAL AND METHODS Forty-one strains of Escherichia coli iso-lated from diarrheal stools of adults and chil-dren were sent to us by the Central Labora-tor:y of the Facul~y of Medical Sciences, Uni-versiey of Campinas, SP, Brazil. The microorganisms were isolated and biochemically classified as recommended by EDWARDS & EWING 4, and serological clas-sification, when carried out, was done by sli-de agglutination tests using OB antisera pur-chased from Wellcome Reagents Ltda. The following monovalent antisera were used: 026B6, 055B5, 0111B4, 0119Bl4, 0125Bl5, 0126B16, 0127B8, 0128B12, 086B7 and 0114K90. Escherichia coli strains to bé ·· examined for enterotoxin production were first cul-tured in nutrient broth incubated at 37º0 for 18 hours. Amounts of 25 ml of Evans medium 6 distributed in 500-ml Erlen-meyer .flaks were inoculated with 1 ml of nu-trient-broth cultures. After incubation àt 37°0 in a rotatory inéubator shaker ande agitated at 135-150 rev/min cultures were spun down at 5000 rpm for ~O minutes. To supernatants carefuHy collected with. a Pasteur's pipette, thimerosal at 1: 10000 was added and the pre-parations so obtained tJ:i.en kept at -20°0 till use. As recommended by DEAN et al.2, infant mice, aged 1-4 êia:ys, in number of four for each E. coli strain w.ere injected .:with~ O .1 rnl of the ST-preparation obtainéd ·•. as .described above. After four hours, the animals ·were sa-crificed and the fluid accumulation ;response quantified by calculating .the ratiQ of the combined weight of the intestine .. of the four mice to the combined weight of the bodies. An intestine to body weight ratio over O. 085 wa, regarded as indicative of enterotoxin pro-duction. Strains which provided positive results in this test were inoculated in 1-liter Erlenmeyer flaks containing 100 ml of Evans' medium and incubated as described for ST-enterotoxin production. Supernatants of these cultures were afterwards 60x concentrated as describ-ed by EVANS et al. 6 and injected in the rabb-it depilated skin to verify if ST-producing strains elaborate also LT. Simultaneousl:y, bacteria-free filtrates obtained as described for LT, without thimerosal, were tested in Yl cells in order to establish wether there was production of an,y LT-toxin detected by this technique 3_ RESULTS Three out of 41 Escherichia coli strains caused an intestine to body weight ratio grea-ter than O . 085 . The presence of a gut that appeared turg-id, swollen and liquid filled was characteris-tic of a positive test allowing usually clear-cut identification of ST-producing strains, as shown in Fig. 1 . 145 SEHAFJM, M. B,: PESTANA DE CAS1'RO. ,\ 1", : R.;\}';C EL. ll. A. & PUSTIGLlONE NETO. L., lwnt-11:tnhl•· ••nt1•rot,,xi n•prNlu('ini,;: 11trainR ui tt-.1•lw r il•hh• 1•011 b1olntP1I f r,nn et1M'J', e,{ dh1rrlwt1, i11 Cmupin<tll, SP. Brazil. Ht-,, Jn.;1. .llo•d . tro1•. ~A<o l'11 1,.1h1 19: l44• 1◄7, 1977. Fig, 1 - kft - lnfanl mou:e,, ino1:uh1\t·d vnth t·vltur,· (iltntt,· e>f a 1wJ?;_,th· .. ~1'·J>t'Odueln,r 1,_::. f'Oli ,:train. Right ln(mll moul!lc ino•nllal•'<I with eultur,, fil1 rnt.- ,ir t! . .-(,li :ctr11ln ~ !<lun,•ln,r dlla-t ,·d lnt,--~th1t•s. Thc s trains 3, 54 and 55 produced in re-peated tcsts means oi 0 .142, 0.123 and 0.122 respectlve)y. The negative stralns produced always vu.lues between 0 .050-0 .059 í21 s trains > and O. 06-0. 069 r 15 s trains/ lollowed by two strains which gave values of O 070 and 0.072 . Putratcs or ST-produclng stralns checked in Yl c~lls did not cause any rounding. Con-centratcd C60xl prepa.rations were unable to elicit an increase of local vascular permeabl-lity 11roving that the studled stralns produc-ed only ST toxln . 1.'he serologlcal classil'ication performed by slide agglutination tests uslng OB antlsera revealed that stralns 3 and 55 belonged to serotype 0128B12. Strain 54 did not aggluti-nate with o.ny a.ntisera. uscd . OISCUSSION Although the occurrence of Escberich ia coli strains which g ive a positive ileaJ loop a.ssay has been already reporteei among us ln 1964 11.1 intormatlon on the frequency and characterization of toxin produced are 146 stlll required. Recently severa! stralns produclng con-comltantly ST and LT toxins have been iso-lated in Sã.o Paulo. Brazil 11, but. in regard to thc isolation oi colibacllll producing only to-xln o! the ST type wc have not lound any re-ports ln thls country. Conslderlng that ln the present work, among 41 stra.ins. three were producing ST toxln, lt might be suggested that this kind oi toxln Is more bwolved in the etiology oi diar-rheaJ diseases caused by E. coli. However i t must be reminded that tcsts for LT produc-tton were not inc1uded ln our routtnal work being carrled out only with lho.se strains which were conflrmed to produce ST tox1n. Anyhow, lt ls apparent lrom the results 3 !7.31%1 positive ST-producing stralns among 41 ch ecked by the lnlant mouse tcst = -that the partlcipatlon of these stralns in in-fantile diarrhea may be important malnly !f we agree that may cases of diarrhea from which collbacilli were isolated mlght be due actually to other causes. SERAFIM, M. B.; PESTANA DE CASTRO, A. F.; RANGEL, H. A. & PUSTIGLIONE NETO, L. - Isolation of heat-stable enterotoxin-producing strains of Esche1·ichia coli isolated from cases of diarrhea in Campinas, SP, Brazil. Rev. Inst. l\Ied. t.-op. São PaJilo 19:144-147, 1977. Another relevant point is that contrarily to LT strains, which frequently are not in-cluded in classical enteropathogenic seroty-pes, tJwo out of three ST-producing strains iso-lated by us belonged to serotype 0128Bl2. Though relative to few strains the data obtained by us ma,y indicate, as it has been pointed out b:y other Authors, that strains in-volved in infantile diarrhea and belonging to known recognized pathogenic sero~ypes when toxigenic produce preferentially if not exclu-sivel:y ST toxin. RESUMO Isolamento de amostras de Escherichia coli produtoras de toxina termoestável de casos de diarréia em Campinas, SP, Brasil Três amostras de Escherichia coli isoladas de casos de diarréia infantil na região de Campinas, SP, demonstraram produzir toxina do tipo ST detectada pelo teste de DEAN. Essas mesmas amostras, quando estudadas na linhagem Yl de tumor de adrenal e na pele de coelhos, não produziram toxina do tipo LT. Em todos os casos as amostras produtoras de toxina ST forneceram no teste de DEAN rela-ções entre o peso dos intestinos/peso do cor-po iguais ou maiores que 0,122. Vinte e uma amostras não produtoras de ST deram rela-ções entre 0,050 e 0,059; 15, entre 0,060 e 0,069 e apenas dois, entre 0,070 e 0,075. ACKNOWLEDGEMENTS Thanks are due to Prof. L.R. Trabulsi for the tests in Yl cells, to the Central Labo-ra tOJ.'.Y of the Faculty of Medical Sciences, University of Campinas, SP, Brazil for sending us the strains of E. coli isolated from cases of diarrheal disease in the town of Campinas, SP and to the Biological Institute, SP for pro-viding us with mice to can;y out the tests. REFERENCES 1. DE, S. N.; BHATTACHARYA, K. & SARKAR, J. K. - A study of the pathogenecity of strains of BacteriJim coli from acute and chronic enteritis. J. Path. Ba<lt. 71: 201-209, 1956. 2. DEAN, A. G.; CHING, Y. C.; WILLIAMS, R. G. & HARDEN, L. B. - Test for -Eschel"ichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honululu. J. In-fect. Dis. 125: 407-411, 1972. 3. DONTA, S. T.; MOON, H. W. & WHIPP, S. C. - Detection of heat labile Escherichia coli ente-rotoxin with use of adrenal cells in tissue culture. Science 183: 344-335, 1974. 4. EDWARDS, D. R. & EWING, W. H. - ldentifi-cation of Ente.-obacteriaceae. 3rd. Ed. New York, Burgess Pub. Co., 1972, 362 p. 5. EVANS, D. G.; EVANS, D. J. & PIERCE, N. F. - Differences in the response of rabbit small in-testine to heat-labile and heat-sta.ble enterotoxins of Esche1·ichia coli. lnfect. & Immun. 7: 873-880, 1973. 6. EVANS, D. J.; EVANS, D. G. & GORBACH, S. L. - Production of vascular permeability factor by enterotoxigenic Escheriehia coli isolated from man. Infect. & Immun. 7: 725-730, 1973. 7. SACK, R. B. - Human diarrheal disease caused by enterotoxigenic Escherichia coli. Appl. Rev. Microbiol. 29: 333-353, 1975. 8. SMITH, H. W. & HALLS, S. - Studies on Es-cherichia coli enterotoxin. J. Path. Bact. 93: 531-543, 1967. 9. SMITH, H. W. & HALLS, S. - Observations by the Jigated intestinal segment and -0ral inocula-tion methods on Eschel'ichia coli infections in pigs, calves, lambs and rabbits. J. Path. Bact. 93: 499-529, 1967. 10. TRABULSI, L. R. - Revelação de colibacilos as-sociados à diarréias infantis pelo método da infec-ção experimental da alça liga.da do intestino do coelho. Rev. lnst. Med. trop. São Paulo 6: 197-203, 1964. 11. TRABULSI, L. R. - Personnal communication. Recebido para publicação em 22/11/1976. 147 

Isolation of heat-stable enterotoxin-producing strains of escherichia coli isolated from cases of diarrhea in Campinas, SP, Brazil

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Isolation of heat-stable enterotoxin-producing strains of escherichia coli isolated from cases of diarrhea in Campinas, SP, Brazil

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