University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln
Uniformed Services University of the Health Sciences U.S. Department of Defense
1982
Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli
Alison D. O'Brien Uniformed Services University of the Health Sciences, [email protected]
Gerald D. LaVeck Uniformed Services University of the Health Sciences
Michael R. Thompson Uniformed Services University of the Health Sciences
Samuel B. Formal Walter Reed Institute of Research
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O'Brien, Alison D.; LaVeck, Gerald D.; Thompson, Michael R.; and Formal, Samuel B., "Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli" (1982). Uniformed Services University of the Health Sciences. 111. https://digitalcommons.unl.edu/usuhs/111
This Article is brought to you for free and open access by the U.S. Department of Defense at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Uniformed Services University of the Health Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. THE JOURNAL OF INFECTIOUS DISEASES * VOL. 146, NO. 6 * DECEMBER 1982
Productionof Shigelladysenteriae Type 1-Like Cytotoxinby Escherichiacoli
Alison D. O'Brien, Gerald D. LaVeck, Fromthe Department of Microbiology,Uniformed Services Michael R. Thompson,*and Samuel B. Formal Universityof theHealth Sciences, Bethesda, Maryland; and theWalter Reed Army Institute of Research, Washington,D.C.
Strainsof Escherichia coli previously implicated or proven to be causesof diarrhea were examinedfor production of a toxinsimilar to thatof Shigella dysenteriae type 1 (Shiga). Organismsgrown in an iron-depletedbroth were lysed by pressure disruption followed by ultracentrifugation.Saline-dialyzed extracts were tested for cytotoxic effects on HeLa cellsthat were neutralizable with antiserum to Shigatoxin. Among the 13 E. coli strainsso analyzed,11 madea Shiga-likecytotoxin in levelsranging from trace (two avirulentisolates) to amountsequivalent to S. dysenteriaetype 1 (two noninvasive strainsthat did notmake E. coli heat-labileor -stableenterotoxins but were isolated frominfants with diarrhea). As withextracts of Shigatoxin, lysates of theseE. coli strainsthat produced high levels of Shiga-liketoxin were enterotoxic for rabbits, paralyticand lethalfor mice, and inhibited protein synthesis in HeLa cells.Thus, these datasuggest that Shiga-like toxin may be anotherheretofore undiscovered factor in the pathogenesisof diarrheacaused by some E. coli strains.
Two mechanismshave been describedby which the microvilliand adhere to the cell membraneof Escherichiacoli can cause diarrhealdisease in hu- intestinalepithelial cells. Invasionof theepithelial mans and animals [1]. Some strainscolonize the cells occursbut apparentlywith less frequencyand small bowel and cause fluidsecretion by elabora- perhaps by a mechanismdifferent from that of tion of heat-labile(LT) and/or heat-stable(ST) shigellae[10-12]. enterotoxins[2-4]. In contrastto these nonin- In a preliminaryreport [13] we indicatedthat vasive, enterotoxicorganisms, certain strainsof some of these LT-negative,ST-negative, S6reny E. coli penetrate and multiplywithin colonic test-negativeorganisms can produce a toxin like epithelial cells [1, 5] and are able to produce thatof Shigelladysenteriae type 1 (Shiga)- thatis, keratoconjunctivitisin guinea pigs (the S6reny the cytotoxicityis neutralizableby antiserumto test) [6]. Infection with these enteroinvasive Shiga toxin-and suggestedthat this toxin could strainsmay cause symptomsthat mimic those seen be responsiblefor the diarrhea-evokingpotential in patientswith shigellosis [1, 5, 7]. That E. coli of thesemicrobes. Subsequently, we had difficulty must be capable of mediatinggastroenteritis by in consistentlyreproducing these findings.How- othermeans is indicatedby the findingthat some ever, we recentlyobserved [14] that the yield of strainsassociated with diarrhealdisease produce Shiga toxincan be increasedif the organismsare no detectableLT or ST and are S6renytest-nega- grownin alkaline mediumor mediumpretreated tive [8-10]. Instead, such organismshave been with the iron-chelatingresin ChelexR (Bio-Rad, shown by electronmicroscopic studies to displace Richmond,Calif.). The purposeof thepresent in- vestigationwas to use these improved culture Receivedfor publication April 30, 1982,and in revisedform methodsto verifythat some E. coli isolatesdo in- July29, 1982. deed produce Shiga-liketoxins. A second interde- The opinionsor assertionscontained herein are theprivate pendentaim of the studywas to determinethe re- viewsof the authors and should not be construedas officialor lationshipbetween the pathogeniccharacter (for as necessarilyreflecting theviews of the Uniformed Services example,enteroinvasive or enterotoxic)of a strain Universityof the Health Sciencesor the Departmentof Defense. of E. coli and its capacityto elaborate Shiga-like This studywas supportedby grantno. R07312from the toxin. UniformedServices University of theHealth Sciences. Pleaseaddress requests for reprints to Dr. AlisonO'Brien, Departmentof Microbiology,Uniformed Services University Materialsand Methods of the HealthSciences, 4301 JonesBridge Road, Bethesda, Bacterial strains. The various bacteria exam- Maryland20814. * Presentaddress: Division of Digestive Diseases, University ined for production of Shiga-like toxin are of CincinnatiSchool of Medicine,Cincinnati, Ohio. describedin table 1. 763 764 O'Brienet al.
Table 1. Characteristicsof bacterial strains examined in a studyof productionof Shigelladysenteriae type 1-like cytotoxin. Culturesupernatant Diarrhea Rabbit Organism, in human ileal Heat-labileHeat-stable Water Sdreny strain(serotype) Source volunteersloop test enterotoxinenterotoxin adsorption* testt Reference Escherichiacoli Laboratorystrain, NT Negative Negative Negative NT Negative 15 K12 strain1133 avirulent (nontypable) HS (nontypable) Healthyadult Negative Negative Negative Negative NT Negative 1, 9 E851/71(0142: Epidemicinfant Positive Negative Negative Negative Positive Negative 9, 16, 17 K86:H6)t diarrhea E74/68(0128: Epidemicinfant Negative Negative Negative Negative Positive Negative 9, 16, 17 K67:H2)t diarrhea 12801/0(0114)t Sporadicinfant Positive Negative Negative Negative NT Negative S diarrhea NC (O125:H21)t Infantdiarrhea NT Negative Negative Negative NT Negative 10 H30 (026)t Infantdiarrhea NT Negative Negative Negative NT Negative 18 S-22-1(0103: Infantdiarrhea NT NT Negative Negative NT Negative 19 H2:K?) RDEC-1(015) Rabbitdiarrhea NT Negative Negative Negative NT Negative 8 H10407 Adultdiarrhea NT Positive Positive Positive Positive Negative 17,20 (078:H11:K2) E2531(O25:K98) Adultdiarrhea NT NT Positive Negative NT Negative 21 TD 514C, Adultdiarrhea NT NT Negative Positive NT Negative 22 (unknown) 4608-58-899 Adultdiarrhea Positive Negative Negative Negative NT Positive 1 (0143) S. dysenteriae Roughavirulent NT Positive Negative Negative Negative Negative 14,23, 24 type1, 60R laboratorystrain Shigellaflexneri Adultdysentery, Positive Negative NT NT NT Positive 1, 25 type2a, M4243 passagedin monkeys Salmonellatyphi- Mousetyphoid, NT Negative NT NT NT Negative 26, 27 murium,W118 virulentin monkeys Pseudomonas Burnpatient NT NT NT NT NT NT None aeruginosa NOTE. NT = nottested. * Inhibitionof wateradsorption in ratjejuna. t Enteroinvasive(causes keratoconjunctivitis in guinea pigs) in Serenytest [6]. t EnteropathogenicE. coli of classicalserotype (026, 055, 086, 0111, 0114, 0125, 0126, 0127, 0128, or 0142). S R. E. Blackand M. M. Levine,personal communication.
Culturemedium. Iron-depletedsyncase broth, distilledwater, and the pH was adjustedto 8.0 brieflydescribed in a previousreport [14], was with2 N NaOH. Next,20 g ofChelex 100 (100-200 preparedas follows.Ten gramsof certifiedcas- mesh;sodium form; Bio-Rad) was addedper liter aminoacids (Difco Laboratories, Detroit), 1.17 g of solution,the broth-resin mixture was incubated of NH4C1,5 g of KH2PO4,5 g of Na2HPO4,and 1 for2 hrat 4 C withstirring, and the iron-chelating ml of tracesalts (5% MgSO4and 0.5% MnC12in beads wereremoved by filtrationthrough a large 0.001N H2SO4)were dissolved in 1 literof quartz- Biichnerfunnel lined withtwo sheetsof filter Shiga ToxinProduction by E. coli 765
paper(Whatman no. 1; ArthurH. Thomas,Phila- A 0. 1-mlaliquot of eachtoxin-serum mixture was delphia).The mediumwas thendistributed into thenadded to HeLa cells.All dilutionswere pre- flasksprecleaned with cleaning solution (RBS-35; paredin Eagle's minimalessential medium with PierceChemical Co., Rockford,Ill.), a surface- Earle'sbalanced salts (HEM Research,Rockville, activeagent that removes iron and othercontami- Md.) plus 2 mML-glutamine, 10% fetalbovine nantsfrom glassware. The broth was sterilized for serum(Flow Laboratories,McLean, Va.), 100 20 minat 121C in an autoclaveand thenallowed units of penicillinG/ml, and 100 ,Agof gen- to cool, and a sterilesolution of 0.2% glucose, tamicin/ml. 0.004% L-tryptophan,and 0.002% nicotinicacid Otheranalyses performed on somebacterial ex- thathad beentreated with 2% Chelexwas added. tractsincluded tests for enterotoxic activity by the To conserveChelex, the resin was regenerated rabbitileal loop assay[30] and assessmentsof the by the followingmethod. The used beads were paralyticand lethal activity for mice [25]. In addi- scrapedfrom the Whatman filter paper, boiled in tion,the technique of Brownet al. was used to 10 volumesof 1 N HC1,and washedfive times in quantitatethe effects of certaincytotoxic extracts distilledwater. The acid-treatedChelex was then on proteinsynthesis in HeLa cells [31]. Finally, boiledin 10 volumesof 1 N NaOH and washed theantigenic relationships among some of the tox- fivetimes in distilledwater, and theexcess water ins containedin extractswere assessedby im- was removedby suctionthrough a frittedglass munodiffusionagainst rabbit antiserum to Shiga funnel. toxin.Immunodiffusion plates were prepared as Preparationof bacterialextracts. Organisms described[23]. wereincubated for 48 hrwith shaking (260 rpm) at 37 C. The sizewas 3 distributedas 2 sample liters, Results litersand 1 literin 4-liter and 2-liter flasks, respec- tively.The bacteriawere harvested by centrifuga- Bacterialcell extractsand culturesupernatants tionat 10,000g at 4 C for20 minand washed from13 strainsof E. coli thathad beengrown in twicein 0.85% NaCl beforethe wetweight was iron-depletedmedium were tested for cytotoxic ef- determined.Weights ranged from 2 to 4.0 g/liter fectson HeLa cellsthat were neutralizable with of medium,and largerbacterial yields generally antiserumto Shigatoxin. These particular isolates indicatedincomplete iron depletion of thebroth wereselected because they were well-characterized and,consequently, reduced toxin production. The andrepresented a pathogenic spectrum that ranged organismswere then resuspended in 20 mlof buf- fromavirulent to virulentby an establisheddiar- fer(0.05 M KC1, 0.01 M MgCl2, and 0.02 M Tris at rhea-evokingmechanism (LT-positive, ST-posi- pH 7.4) and disruptedby two passages through a tive,or enteroinvasive)or by undefinedmeans Frenchpressure cell at 15,000psi. Lysateswere (LT-negative,ST-negative, and Sereny test- thensubjected to ultracentrifugationat 100,000 g negative).Positive controls for the study included for70 minand supernatantswere dialyzed against lysatesand culturesupernatants of S. dysenteriae 0.85% NaCl at 4 C. The proteincontent of these type1 strain60R, whichproduces high levels of extractswas estimatedspectrophotometrically Shiga toxin[14, 23, 24], as well as a strainof [28]. Shigellaflexneri type 2a, whichproduces Shiga- Biologicand immunologicassays. Cytotoxici- like toxin[25]. Two otherbacterial species were tytests were performed with HeLa cells(Walter includedin thisinvestigation: Salmonella typhi- Reed ArmyInstitute of Research,Washington, murium(another Enterobacteriaceae) and the D.C.) by the methodof Gentryand Dalrymple unrelatedgram-negative microbe, Pseudomonas [29]. Neutralizationtests were done by mixing aeruginosa.Some pertinentcharacteristics of equal volumesof seriallydiluted rabbit antiserum these microbesare listedin table 1. to Shiga toxin or preimmunizationrabbit serum The resultsof the cytotoxicityevaluations are with10 50% cytotoxicdoses of toxincontained in shown in table 2. The organismswere arbitrarily filteredbacterial extracts and thenincubating the groupedinto fourcategories according to the ef- mixturesfor 1 hr at 37 C, followedby overnight fectsof the bacterialextract on HeLa cells. Cate- incubationat 4 C. Rabbit antiserumto Shiga toxin gory 1 contained those isolates that made cell- was elicitedagainst purified Shiga toxin [23] by a associatedcytotoxins that were not neutralizedby previouslydescribed immunization protocol [23]. rabbitantiserum to Shiga toxin,such as P. aeru- 766 O'Brienet al.
Table 2. Productionof cytotoxins,categorized by similarityto the toxinof Shigelladysenteriae type 1 (Shiga), by variousbacterial strains. Cytotoxicityfor HeLa cells Cytotoxicity CD50/ml CDso/mg neutralizedby rabbit of culture of proteinin antiserumto Shiga Category Toxin production Bacteria supernatant bacterialextract toxin(titer)*
1 Cytotoxinnot Shiga-like Escherichiacoli E2531 ND 9 No NC ND 15 No Pseudomonasaeruginosa ND 60 No 2 Trace levels of Shiga-like E. coli toxin (<10 CD50/mgof K12 (strain1133) ND 2 Yes (1:3,200) bacterialextract) HS ND 10 Yes (1:6,400) 3 Low to moderatelevels of Salmonella typhimurium Shiga-liketoxin W118 ND 20 Yes (1:6,400) E. coli TD 514C, ND 20 Yes (1:6,400) RDEC-1 ND 40 Yes (1:12,800) 460-8-58-899 ND 40 Yes (1:6,400) E74/68 ND 60 Yes (1:6,400) E851/71 ND 100 Yes (1:6,400) H10407 ND 100 Yes (1:3,200) 12801/0 ND 200 Yes (1:1,600) Shigellaflexneri type 2a (strainM4243) 50 300 Yes (1:3,200) 4 High levels of Shiga or E. coli Shiga-liketoxin H30 1 x 103 1 x 104 Yes (1:800) S-22-1 1 x 104 4 x 104 Yes (1:3,200) S. dysenteriaetype 1 1 x 104 3 x 105 Yes (1:1,600-1:6,400) (strain60R) = = NOTE. CDSo cytotoxicdoses requiredto kill 50% of HeLa cells; ND not detected. * Highestdilution of serumthat completely protected HeLa cells from10 CD5oOf toxin. ginosa.The microbesassigned to category2 (trace [18]. Theyreported that culture filtrates of the levelsof Shiga-likecell-associated cytotoxin) in- bacteriumwere cytotoxic for Vero cells. The other cludedthe two avirulent E. coli strains1133 [15] organism,strain S-22-1, was obtainedfrom M. andHS [1,9]. Theagents listed in category 3 (pro- Gurwith(Michigan State University, East Lansing). ducersof low to moderatelevels of Shiga-like Gurwithand Williams [19] observed that a culture cytotoxins)included seven strains of E. coli rang- filtrateof thestrain, which was isolatedfrom the ingfrom an ST-positivestrain that produced low stool of a childwith diarrhea, grown in brain- levelsof toxin(TD 514C) [22]to an enteropatho- heartinfusion broth was cytotoxic for HeLa cells. genicstrain (12801/0) that made nearly as much Thatthe extracts from these two LT-negative, ST- toxinas S. flexneritype 2a. We foundthat S. ty- negative,Sereny test-negative strains of E. coli phimuriumstrain W118 [26, 27] also made low isolatedfrom infants with diarrheal disease [18, levelsof a cytotoxin,an observationthat supports 19] do indeedmake a toxinsimilar to Shigatoxin thefindings of others(F. C. W. Koo and J.Peter- was verifiedby otherbiologic and immunologic son, personalcommunication). The relationship tests.Lines of identitywere seen when extracts of betweenthe S. typhimuriumcytotoxin described E. colistrain H30, E. colistrain S-22-1, and S. dys- by theseinvestigators and the Shiga-liketoxin enteriaetype 1 strain60R werediffused against describedin thepresent investigation remains to rabbitantiserum to Shiga toxin(figure 1). Fur- be determined. thermore,cell extractsof thesetwo strainsof Category4 includedtwo strains of E. coli that E. coli, like S. dysenteriaetype 1 strain60R, made levelsof Shiga-liketoxin equivalent to the paralyzedand killedmice, inhibited protein syn- strain60R positivecontrol. One of themicrobes, thesisin HeLa cells,and causedfluid accumula- strainH30, was describedby Konowalchuk et al. tionin ligated rabbit ileal loops (table 3), as didfil- ShigaToxin Production by E. coli 767
Figure1. Resultsof immunodiffu- sionof rabbit antiserum to toxinpro- duced by Shigelladysenteriae type 1 (centerwell) againstcrude cell ex- ' ' tractsof S. dysenteriaetype 1 strain t~,.::." 60R (well A), Escherichiacoli strain H30 (well B), and E. coli strain S-22-1(well C).
tered culturesupernatants of the microbes(data among such isolates toxic activityvaried as much not shown). By contrast,an extractof E. coli as 10,000-fold.The basis forthe diversity in yields strain12801/0 inhibited protein synthesis in HeLa of cell-associatedtoxin activity was not examined cells but was not lethalfor mice or enterotoxicfor but parallels the situationseen among species of rabbits,and an extractof E. coli K12 strain1133 shigellae- for example, S. dysenteriae makes was negativefor all threeactivities. It should be 104-105more cytotoxicmaterial per milligramof noted that the mouse lethalityand rabbitentero- cell extractprotein than does S. flexneri[14, 25]. toxicityassays forpurified Shiga toxinare at least Several theoriesmay be proposed to explain the 104times less sensitivethan are testsfor cytotoxi- E. coli strain-dependentdifferences in elaboration cityor inhibitionof proteinsynthesis [14, 32]. of Shiga-like toxin. (1) The regulatorymecha- nisms that control toxin production by the be distinct.For a Discussion organismsmay quite example, category4 (high-levelproducer of toxin)organism The presentstudy established that some strainsof may make toxin constitutively,whereas toxin E. coli make a Shiga-liketoxin and showed that levels made by E. coli in category2 or 3 (trace to
Table 3. Otherbiologic properties of extractsof representativebacteria in a studyof bacterialcytotoxins. Lethalityfor mice Inhibitionof protein Enterotoxicityfor (LDso/,4g synthesis(ID50/mg Bacteria rabbits* of protein)t of protein)l Shigelladysenteriae type 1, strain60R Yes Yes (50) Yes (105) Escherichiacoli S-22-1 Yes Yes (60) Yes (105) H30 Yes Yes (90) Yes (105) 12801/0 No No Yes (90) K12, strain1133 No No No (<10) * Extracttested at a dilutionof 1:20. St Hind leg paralysisobserved before death of mice. IDo50= dose requiredto inhibitprotein synthesis in HeLa cells by 50%. 768 O'Brienet al.
moderatelevels of toxin production) may be deter- Shiga-liketoxins was notexamined in thisinvesti- minedby a repressibleor inducible mechanism. (2) gation.The factthat Shiga-like toxic activity can Toxinmade by category 2 or 3 microbesmay vary be neutralizedby antiserum to purifiedShiga tox- structurallyfrom that producedby category4 in suggestssome degree of homologybetween the bacteria.Perhaps structural differences translate toxins.Other data that support the hypothesis that to variabilityin biologicactivities. (3) Category2 the moleculesare physicallysimilar include the and 3 bacteriamay manufacture large amounts of rangeof biologic activities of the two moieties and biologicallyinactive toxin that compete with ac- theimmunodiffusion results. Shiga toxin appears tivetoxin in bioassays. If thereason for variability to be composedof two subunits: an enzymatically in toxicityamong strains of E. coli is thesame as activeA subunit(-30,500 daltons)that when thatfor shigellae, the last postulateis not likely nickedand reducedinactivates 60S ribosomesand becausea previousinvestigation [14] showed that inhibitspeptide chain elongation and six or more no biologicallyinactive toxin was foundin ex- copies of a B subunit(-5,000 daltons)that is tractsof S. flexneri.Genetic and biochemicalex- presumedto functionin the binding of toxin to the perimentsshould address this issue of variability targetcell [34, 35]. Thus,Shiga-like toxins prob- inthe quantity of Shiga-liketoxin made by isolates ably containone or bothof thesecomponents. of E. coli. Purificationof the toxicmaterial from E. coli Two lines of circumstantialevidence suggest strainH30 shouldclarify this issue. thatthe Shiga-liketoxin may play a role in the pathogenesisof some cases of E. coli-conferred diarrhea.(1) Two strainsof E. coli implicatedas References mediatorsof gastroenteritisin children made high levelsof toxinbut did not elaborate LT 1. DuPont,H. L., Formal,S. B., Hornick,R. B., Snyder, Shiga-like M. J. D. E. or ST and were The J.,Libonati, P., Sheahan, G., LaBrec, H., S6renytest-negative. (2) Kalas, J. P. Pathogenesisof Escherichiacoli diarrhea. avirulentE. coli strains1133 and HS madeonly N. Engl.J. Med. 285:1-9,1971. traceamounts of toxin.This finding suggests that 2. Gyles,C. L. Heat-labileand heat-stableforms of the if a Shiga-liketoxin is in facta virulencedetermi- enterotoxinfrom E. coli strainsenteropathogenic for then eitherthese isolatesdo not make pigs.Ann. N.Y. Acad. Sci. 176:314-322,1971. nant, C. D. A. A heat-labileenterotoxin toxinto be detrimentalor anotherfactor 3. Gyles, L., Barnum, enough fromstrains of Escherichiacoli enteropathogenicfor or factorsmay be requiredto conferpathogenici- pigs.J. Infect.Dis. 120:419-426,1969. ty. Such a factorcould be a mechanismof 4. Smith,H. W., Gyles,C. L. Therelationship between two adherenceto theintestinal mucosa; indeed, Smith apparentlydifferent enterotoxins produced by entero- and establishedthat pathogenicstrains of Escherichia coli of porcineorigin. Linggood [33] clearly 1970. strainsof E. thatlack the K88 J. Med. Microbiol.3:387-401, enterotoxigenic coli 5. Ogawa, H., Nakamura,A., Sakazaki, R. Pathogenic colonizingfactor fail to causediarrheal disease in propertiesof "enteropathogenic"Escherichia coli from pigs. That some LT-positive,ST-positive, or diarrhealchildren and adults.Jpn. J. Med. Sci. Biol. enteroinvasivestrains of E. coli also madelow to 21:333-349,1968. moderateamounts of toxin 6. Sbreny,B. Experimentalshigella keratoconjunctivitis; a Shiga-like (table 2) Sci. a toxinin preliminaryreport. Acta Microbiol.Acad. Hung. does not ruleout role forShiga-like 2:293-296,1955. disease caused by E. coli. Rather,enterotoxic 7. Sakazaki,R., Tamura,K., Saito, M. Enteropathogenic strainssuch as E. coli strainH10407 may colonize Escherichiacoli associated with diarrhea in children and a regionof theintestine where Shiga-like toxin is adults.Jpn. J. Med. Sci. Biol. 20:387-399,1967. ineffective. toxin ac- 8. Cantey,J. R., Blake,R. K. Diarrheadue to Escherichia Alternatively,Shiga-like may coli in therabbit: a novelmechanism. J. Infect.Dis. tivelycontribute to the developmentof clinical 135:454-462,1977. manifestationsin E. coli strainH14047-induced 9. Levine,M. M., Berquist,E. J.,Nalin, D. R., Waterman, diarrhea. Obviously,further studies are required D. H., Hornick,R. B., Young,C. R., Sotman,S., to definethe role (if any) of Shiga-liketoxin in the Rowe,B. Escherichiacoli strainsthat cause diarrhoea of diarrhea.These could but do not produceheat-labile or heat-stableentero- pathogenesis investigations toxinsand arenon-invasive. Lancet 1978. includean examinationof whetherthe toxin func- 1:1119-1122, 10. Ulshen,M. H., Rollo,J. L. Pathogenesisof Escherichia tions in vivo as an enterotoxinor as a cytotoxin. coli gastroenteritisin man-anothermechanism. N. The structuralrelationship between Shiga and Engl.J. Med. 302:99-101,1980. ShigaToxin Production by E. coli 769
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