Colonization factors andEscherichia coli belonging to enterotoxin-associated serotypes

Hemagglutination (HA) tests using human and bovine erythrocytes and microagglutination tests using pili-specific antisera (PSA) were performed to examine 168 strains ofEscherichia coli belonging to enterotoxin-associated serotypes for colonization factors (CFs). Seventy-one (42%) of these 168 strains possessed at CF, but only 10 (6%) were found positive by both HA and PSA tests. Groups of test strains from different sources (feces, urine, blood, and wounds) were not found to contain statistically different percentages of CF-positive strains. Strains producing heat-stable enterotoxin alone were less frequently associated with a CF than were other enterotoxigenic and nonenterotoxigenic strains. Strains showing heat-labile hemolytic activity and belonging to serotype O6: H—were less likely (P=0.014, Fisher's exact probability) to contain a CF than were similarly hemolytic strains belonging to other serotypes.     

Plasmids coding for heat-labile enterotoxin production isolated from Escherichia coli O78: comparison of properties.

Nineteen enterotoxigenic Escherichia coli strains of serogroup O78, isolated in different geographical areas from humans with diarrheal diseases, were tested for their ability to transfer enterotoxin production. All of the strains originally produced heat-labile enterotoxin, and 16 also produced heat-stable enterotoxin and colonization factor antigen I. Plasmids coding for the production of heat-labile enterotoxin only were transferred from 13 strains. Some properties of these plasmids were compared. All were fi+, but they could be divided into three groups on the basis of their incompatibility reactions, ability to restrict E. coli K-12 phages, and size. The three heat-labile enterotoxin plasmids isolated from African strains all belonged to one enterotoxin plasmid group. The heat-labile enterotoxin plasmids from the Asian strains were divided into two groups, those from serotype O78.H11 differing from those from serotype O78.H12.     

Cytolethal distending toxin (CLDT) production by enteropathogenic Eschrichia coli (EPEC)

Culture supernates of 16 strains of EPEC belonging to 6 different serogroups, when assayed on Chinese Hamster Ovary (CHO) cells up to 96-120 h, induced distinct morphological changes. The supernate activities were heat-labile, unrelated to heat-labile enterotoxin (LT), verotoxin (VT), or hemolysin, did not show necrosis in rabbit skin and caused no fluid secretion in the rabbit ileal loop assay (RILA). Simultaneous production of CLDT and heat-stable enterotoxin (ST) were detected in four EPEC strains.     

Production of enterotoxins by strains of Escherichia coli isolated from pigs

The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO. Vero and Hela cells and ILT. More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains). No correlation between intensity of ILT and particular serotype was observed. Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin. The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs. Strains of E. coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes. The cytotoxic activity of supernatants of E. coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed. This suggests the production by the strains of enterotoxin LT and cytotoxin VT. Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs. This suggests the production by these isolates of a toxin (toxins) differing from the E. coli enterotoxins.     

Heat-Stable Enterotoxin Produced by Yersinia enterocolitica Isolated from Patients

Twenty-three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15-fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 μg of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C × 20 min or 100 C × 60 min). These observations indicate that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat-stable enterotoxin of Escherichia coli. However its physicochemical properties seem to be different from those of E. coli.     

Susceptibility of the Mouse Intestine to Heat-Stable Enterotoxin Produced by Enteropathogenic Escherichia coli of Porcine Origin

Ligated intestinal loops of mice were found suitable for the assay of heat-stable enterotoxin produced by enteropathogenic Escherichia coli strains of porcine origin; loops inoculated with heat-labile enterotoxin failed to react.     

Enterotoxigenicity of diarrhoeal isolates of Salmonella bareilly

Whole cell cultures, cell-free supernatants, and cell sonicates from ten strains of Salmonella bareilly induced fluid accumulation in ligated rabbit and rat ileal loops. All strains had an intracellular vascular permeability factor, half were suckling mouse positive indicating the presence of a heat-stable type of activity. The toxin(s), however, were Immunologically distinct from the heat-labile toxin of Escherichia coll LT and cholera toxin. Besides enterotoxigenicity, all strains exhibited potential Invasive character.The authors are with the Department of Microbiology, Panjab University, Chandigarh, India 160 014. M. Saxena is the corresponding author.     

Somatic O Antigen Relationship of Brucella and Vibrio cholerae

The antigenic relationship between Brucella species and Vibrio cholerae was examined by agglutinin and agglutinin-absorption tests by using rabbit antisera. Brucella antisera agglutinated only the Inaba serotype of V. cholerae and at low titer. Inaba-reactive antibody was absorbed by either heat-stable (100 C, 2 hr) Ogawa or Inaba O antigens. Cholera antisera from rabbits immunized with either O or HO antigens of either Ogawa or Inaba serotypes contained brucella agglutinins. This activity was absorbed completely from Ogawa antisera by either Ogawa or Inaba O antigens but only partially from Inaba antisera by Ogawa O antigen. These findings support the claim of Gallut that the cross-reaction is due to heat-stable O antigens of V. cholerae rather than heat-labile flagellar antigens as described in many text books. The cross-reactive component is more dominant in the Inaba than in the Ogawa serotype of V. cholerae.     

Pilus antigen 987P produced by strains of Escherichia coli serotypes O141:K--, H- and O8:K85:H--

Escherichia coli strains isolated from the alimentary tract of 68 weaned and 44 unweaned pigs with diarrhoea in various parts of Hungary, were tested for the presence of pilus antigens K88, K99 and 987P. K88 was detected in 30% of the strains from newborn pigs and in 12% of the strains isolated from weaned pigs. One strain carried K99. Based on agglutination test and immunoelectron microscopic studies with specific absorbed antisera, five non-haemolytic E. coli strains isolated from newborn pigs were found to produce so-called 987P pili. Three of these strains were designated serologically as O8:K85:H--,987P+ and two as O141: K--:H--,987P+. The Y1 cell assay, the infant mouse assay, and the ligated intestinal loop assay in less than 3-week-old pigs indicated that none of the strains produced heat-labile enterotoxin but all produced a heat-stable enterotoxin detectable in infant mice and in pig loops (STa). All the strains induced diarrhoea in newborn, colostrum deprived pigs and colonized the lower small intestine by adhesion to the villous epithelium. The results have confirmed earlier findings about adhesive virulence attributes caused by 987P pili.     

Identification of Heat-Stable Enterotoxin-Producing Strains of Yersinia enterocolitica and Vibrio cholerae Non-O1 by a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay

Using a mouse monoclonal antibody (MAb) 2F raised against Vibrio cholerae non-O1 heat-stable enterotoxin (NAG-ST) which also recognizes a shared epitope of Yersinia enterocolitica heat-stable enterotoxin (Y-ST), a competitive enzyme-linked immunosorbent assay (ELISA) was developed for independent detection of NAG-ST and Y-ST. There was good concordance between the Y-ST ELISA and the suckling mouse assay (SMA) for detection of Y-ST from test strains of Y. enterocolitica, and the Y-ST ELISA can effectively replace the SMA for routine detection of Y-ST. On the contrary, evaluation of the NAG-ST ELISA and the SMA using 139 strains of V. cholerae non-O1 showed discordant results and this was attributed to the presence of the suckling mice active factor(s) such as El Tor hemolysin and to the production of low amounts of NAG-ST. Concentration of culture supernatants of V. cholerae non-O1 followed by heating at 100 C was essential to obtain reproducible results by both the NAG-ST ELISA and the SMA. The ELISA developed in this study can be used for the identification of biologically active strains. While recently genetic methods such as polymerase chain reaction became available and were very reliable and simple techniques, the ELISA in this study has an advantage in detecting biologically toxic gene products of the strains. The genetic methods cannot differentiate silent STa genes which we often encounter in the case of Y. enterocolitica.     

Response of Chinese hamster ovary cells to a cytolethal distending toxin (CDT) of Escherichia coli and possible misinterpretation as heat-labile (LT) enterotoxin

Increased incubation of the Chinese hamster ovary cell (CHO) assay up to 96–120 h allowed differentiation of cytotonic and cytotoxic effects. Strains of Escherichia coli O128 found to produce a heat-labile cytolethal CHO toxin were compared with E. coli heat-labile enterotoxin (LT). The cytolethal toxin was unrelated to LT, heat-stable enterotoxin (ST), Verotoxin (VT) or hemolysins. Since CHO elongation induced by either the E. coli LT or E. coli O128 filtrates could not be differentiated after 24 h, continued incubation for 96–120 h was essential for observation of progressive morphological changes and cytolethal events. Comparative responses in at least two cell systems is recommended to prevent misinterpretation of elongation at 24 h in the CHO assay.     

Acquisition and maintenance of enterotoxin plasmids in wild-type strains of Escherichia coli

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.     

Properties of wild-type strains of enterotoxigenic Escherichia coli which produce colonization factor antigen II, and belong to serogroups other than O6

Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than O6 and produced colonization factor antigen II, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransferring plasmid, NTP165, from a strain of E. coli O168. H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTP165 depended on the recipient strain: a biotype A strain of serotype O6. H16 expressed CS1 and CS3; a biotype C strain of serotype O6. H16 expressed CS2 and CS3; strain K12 and strain E19446 of serotype O139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype O139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lost the plasmid coding for CS antigens produced both CS1 and CS3 after the introduction of NTP165.     

Escherichia coli Constructs Expressing Human or Porcine Enterotoxins Induce Identical Diarrheal Diseases in a Piglet Infection Model

To develop a piglet model for studying diarrheal disease and developing vaccines, we challenged gnotobiotic piglets with isogenic Escherichia coli strains constructed to express porcine 987P(F6) fimbriae and a heat-labile or a heat-stable enterotoxin to examine clinical outcomes. Piglets developed identical diarrheal diseases when inoculated with constructs expressing human or porcine enterotoxins.     

Characterization of enterotoxin produced by four Yersinia enterocolitica strains of pig origin

All four isolates of Yersinia enterocolitica and one isolate of Y. frederiksenii from pigs were found to be enterotoxigenic. Whole-cell preparations of Y. enterocolitica isolates did not induce any change in the rabbit ligated gut test after 6 and 18 h of inoculation, but Y. frederiksenii on the other hand showed a positive gut response at 18 h. Cell-free supernatant (CFS) of all five isolates induced dilatation in the rabbit gut up to 6 h, after which Y. enterocolitica became negative, while Y. frederiksenii continued to show a reaction up to 18 h. CFS of all five isolates were also found positive with the infant mouse test. Of the five isolates of Yersinia, three gave a positive reaction for the permeability factor on rabbit skin. Yersinia enterotoxin could be concentrated by methanol extraction. It was stable at 100°C for 20 min and at 120°C for 15 min. However, its activity was lost at low (2.0) and high pH (10.0). Enterotoxic preparations of Y. enterocolitica lost part of their enterotoxic activity upon dialysis.     

Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo

Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals.

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

Detection and identification of E. coli producing heat-labile enterotoxin type I by enzymatic amplification of a specific DNA fragment

The polymerase chain reaction (PCR) was used to identify strains of Escherichia coli which produce heat-labile toxin type I (LTI). Amplification primers were designed to detect E. coli strains of human as well as porcine origin. This assay was used to test the ATCC 37218 strain, which carries a recombinant plasmid with the genetic information for production of porcine LTI (pLTI). In addition, three clinical E. coli isolates of human and one of porcine origin were tested. All clinical isolates were reported to produce heat-labile enterotoxin (hLTI and pLTI, respectively) when tested by the Y1 adrenal cell method and/or by the CHO cell method. All strains yielded the expected 275 bp DNA fragment after enzymatic amplification. This fragment was further identified by allele specific oligonucleotide hybridization. Alternatively, the fragment was identified by a SmaI restriction enzyme site which is present in the genes of both the E. coli isolated from humans and pigs. The detection limit determined in water with the ATCC 37218 strain was 20 bacteria. The amplified sequence included a CfoI polymorphism which allowed to distinguish between the genes coding for pLTI and hLTI. All of the strains tested showed this polymorphism as expected. Depending on the identification method chosen, SmaI digestion or oligonucleotide hybridization, pure water can be analysed within 8 h or 12 h, respectively. This method may be adapted to environmental and food samples.     

Production of heat-stable enterotoxin II by chicken clinical isolates of Escherichia coli

Abstract Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat-stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat-stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100°C and was neutralized by anti-heat-stable enterotoxin II antiserum. Purified heat-stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII-producing E. coli is implicated in chicken diarrhea.