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.     

Identification of enterotoxigenic staphylococci from sheep and sheep cheese

The total of 127 Staphylococcus aureus strains obtained from sheep and sheep cheese were examined for their biochemical activities, biotypes, phage patterns, and ability to produce enterotoxins. Of the 83 staphylococcal strains isolated from animals 77 (93%) were classified as the C biotype. Of this group of sheep-adapted strains, 61 (79%) were sensitive to phage 78, and 46 (60%) produced enterotoxin C exclusively. The three isolated belonging to the A biotype produced enterotoxin D, and two of the three unclassifiable strains produced enterotoxin A. Of the 44 staphylococcal strains isolated from sheep cheese, there were 37 (84%) identified as the C biotype. From this series, 31 (84%) strains were lysed with phage 78, 6 (16%) strains produced enterotoxin C, and 1 strain produced enterotoxin A. One of the six strains determined as the A biotype produced enterotoxin D. C biotype strains, especially of ovine origin, are an exception among animal staphylococci, because a large number of them are enterotoixgenic. The C antigenic type is the most usual of the known enterotoxins in staphylococci of animal provenance.     

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.     

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis

Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning. Microbiological Societies.     

Enterotoxin Production by Lecithinase-positive and Lecithinase-negative Clostridium perfringens Isolated from Food Poisoning Outbreaks and other sources

Strains of Clostridium perfringens from a variety of sources were examined for their ability to produce enterotoxin in vitro. Fifty-six of 65 (86%) strains isolated from separate outbreaks of food poisoning were found to be enterotoxigenic, only two of 174 strains from other sources produced enterotoxin. The ability to produce this toxin was not confined to particular serotypes: types frequently encountered as the cause of outbreaks were also isolated as enterotoxin-negative strains from faeces, minced beef and meat carcasses. Loss of toxigenicity was also observed in different serotypes. Five strains of lecithinase-negative Cl. perfringens produced high levels of enterotoxin. Four strains of Clostridium plagarum failed to produce enterotoxin although they were serologically typable with the Cl. perfringens antisera.     

Production of cholera-like enterotoxin by Aeromonas hydrophila

A total of 249 strains of mesophilic Aeromonas including 179 A. hydrophila and 70 A. caviae were tested for production of cholera-like enterotoxin by reversed passive latex agglutination (RPLA) assay. A cholera-like enterotoxin neutralized with cholera antitoxin was demonstrated in the culture filtrates from eight (4.5%) of the 179 A. hydrophila strains, while none of A. caviae strains revealed the enterotoxin production in the test. Production of the cholera-like enterotoxin in the eight strains of A. hydrophila was also confirmed by enzyme-linked immunosorbent assay (ELISA).     

The Biochemical Activity of Enterotoxin and Non-Enterotoxin Producing Staphylococci

One hundred and sixty-nine staphylococcal strains of human origin have been tested for production of enterotoxin A, B or Ci, coagulase activity, DNase activity, typical growth on ETGP-agar, hemolysin production and the breakdown of mannitol under aerobic conditions. Very good correlation was observed between enterotoxin production and coagulase activity, in that 82 % of the enterotoxin producing strains also synthesized coagulase. The correlation between DNase activity and positive reaction in mannitol to enterotoxin production was also good (80 % of the enterotoxic strains produced both DNase and aerobic acid from mannitol). Of the enterotoxin producing strains 66 % hemolysed bovine erythrocytes and 61 % were ETGP-positive. However, the frequency of hemolysing respectively ETGP-positive but non-enterotoxin producing strains was very high, viz. 46 % respectively 32 %. It is concluded that enterotoxin production can not to a satisfactory degree of security be predicted by means of the other biochemical characters.     

Identification of a Fourth Staphylococcal Enterotoxin, Enterotoxin D

A fourth staphylococcal enterotoxin was identified serologically with antiserum to the very crude enterotoxic products of growth of a strain which also produces enterotoxin C, and then with antiserum to the considerably purified enterotoxic antigen of a strain which produces only the new enterotoxin. The identification of this antigen as enterotoxin D was based on the following observations. It was produced by strains which do not produce enterotoxins A, B, or C; it was absent in the growth products of nonenterotoxigenic strains; when appreciably purified, it was associated with emetic activity in the cat, and its biological activity was neutralized only by antisera containing its specific antibody and not by antibodies to enterotoxins A, B, and C. Staphylococcal strain 494 (ATCC 23235) was selected as the prototype strain. The production of this enterotoxin alone and together with enterotoxin A by strains of food-poisoning origin indicates that its role in food poisoning is second in frequency only to that of enterotoxin A. The incidence of production of enterotoxins A, B, C, and D, and of unidentified cat emetic substances by strains from several source categories, is presented.     

Evidence for a further enterotoxin complex produced by Bacillus cereus

Abstract Out of 321 strains of Bacillus cereus from several sources and isolated in four different countries, 239 (74%) produced cytotoxins. Only 127 (53%) of the cytotoxic strains were positive for the B-component gene of the haemolysin BL (enterotoxin) by polymerase chain reaction (PCR). Western blots using antiserum produced against enterotoxin(s) gave positive results for 199 (83%) of the cytotoxic B. cereus strains. On closer examination of seven of the strains, involved in food poisoning, we found that two strains completely lacked the L₂- and B-components (of the haemolysin BL), and two strains were negative for the B-component gene by PCR, but were positive for the L₂-component. From our experiments we concluded that there is at least one enterotoxin complex in addition to the haemolysin BL enterotoxin and enterotoxin T.     

Enterotoxin formation by Clostridium perfringens type A studied by the use of fluorescent antibody.

Fluorescein isothiocyanate-conjugated antibody to purified enterotoxin of Clostridium perfringens was used to study the intracellular formation of enterotoxin by this organism. Enterotoxin was detected at 4 h of growth at the end of the cell containing forespore. With the development of the spore, enterotoxin accumulation continued and involved the entire length of the cell until its lysis with the release of enterotoxin and mature spore. The spores did not contain demonstrable enterotoxin. Only a certain number of the sporulated cells of the enterotoxigenic strains studied produced this toxin. The amount of enterotoxin produced varied with sporulation percentage, and between strains and individual cells.     

The detection of a choleriform thermolabile enterotoxin in clinical strains of Proteus isolated in different infections

The capacity of Proteus strains, isolated from patients with purulent inflammatory, urological and enteric infections, for the production of choleriform thermolabile enterotoxin was studied by means of the enzyme immunoassay (EIA) with the use of antitoxic serum to Escherichia coli enterotoxin. Out of 125 strains, 27 (21.6%) showed the capacity for producing choleriform thermolabile enterotoxin in EIA experiments. The results thus obtained indicate that EIA techniques can be used, in principle, for detecting the capacity of Proteus for the production of choleriform thermolabile enterotoxin.     

Enterotoxigenicity of Salmonella strains of various origins

The study of the enterotoxigenicity of S. typhimurium with the use of the skin test on rabbits (to detect the delayed permeability factor) has revealed that these strains produce an enterotoxin similar to Escherichia coli thermolabile enterotoxin (TLE). Study of the enterotoxic activity of lysates obtained from 39 S. typhimurium strains and 5 S. dublin strains by sonication has revealed that 87% of S. typhimurium strains and all S. dublin strains produce an enterotoxin similar to E. coli TLE, as demonstrated by all tests used in this investigation, while 59% of S. typhimurium cultures and all S. dublin strains have been positive when tested for the capacity of producing the rapid permeability factor. "Hospital" strains and polyresistant cultures isolated from the environment (phagovar 20) are characterized by a higher rate of producing an enterotoxin similar to E. coli TLE, detected by the tests used in this investigation (90%), than antibiotic-sensitive strains of different origin (78%).     

苏云金芽胞杆菌肠毒素基因的PCR检测

采用多重引物PCR进行了 45株苏云金芽胞杆菌、2株蜡状芽胞杆菌和 2株球形芽胞杆菌溶血素BL ,肠毒素T和entS基因的检测 ,结果表明 95 6%苏云金芽胞杆菌含溶血素hblA基因 ,91 1 %含bceT基因 ,93 3%含entS基因。用两种商业化肠毒素检测试剂盒TECRA和RPLA进行所有菌株肠毒素的体外免疫测定 ,大部分苏云金芽胞杆菌和阳性蜡状芽胞杆菌都能产生不同水平的肠毒素活性 ,同hblA基因PCR检测结果基本相符。尽管DBT0 0 7和T2 4 0 0 1含有hblA基因 ,但用TECRA却检测不到肠毒素 ;Dmu39菌株不含肠毒素基因 ,但用TECRA却检测出高的肠毒素活性。苏云金芽胞杆菌BDT2 4 8和球性芽孢杆菌不含肠毒素基因和肠毒素。结果表明昆虫病原菌苏云金芽胞杆菌的安全性有待进一步研究     

Detection of staphylococcal enterotoxigenicity IV. Strains isolated in 1981 and 1982

Enterotoxin A, B, C, D and E detection and typing was undertaken in 807 staphylococcal strains isolated from food, breast milk, clinical material, diarrhoeal stools and hospital-collected swabs in 1981 and 1982. One hundred and sixty-six of the strains produced enterotoxin, most frequently type A or C, less so type D or B. There were single instances of strains with double toxin production: AB, AC or AD. Nine hundred and ten supernatants collected in 1972-1973 were additionally tested (after a lapse of 8 years) for type D enterotoxin; there were 152 positive specimens, predominantly relating to strains isolated from tinned cocoa and delicatessen, with 26 of the supernatants containing AD and BD enterotoxin combinations. For the first time the authors' laboratory detected strains producing enterotoxin F and the combination.     

Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407

Abstract The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E. coli strains EWD 299 and H 10407. The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain. Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to those of the human heat-labile enterotoxin from strain H 10407. However, the patterns of plasmids from the 21d and H 10407 strains were different. The 21d strain had no band corresponding to the 42-MDa plasmid of the H10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid. These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H10407 strain.     

Biological characteristics of an enterotoxin produced by Bacillus cereus.

An enterotoxin synthesized during exponential growth by Bacillus cereus produces fluid accumulation in rabbit ileal loops, alters vascular permeability in the skin of rabbits, and kills mice when injected intravenously. All activities are eluted simultaneously from a Sephadex G-75 column and are distinct from the hemolysin and egg yolk turbidity factor of B. cereus. The enterotoxin is a true exotoxin. It interacts with intestinal receptor sites in a highly transient manner in the ileal loop system. Rabbit immune serum produced against the culture fluids from one strain of B. cereus neutralized the three biological activities in all other strains tested except strain B-6-ac for which none of the activities were neutralized. Enterotoxin proved to be unstable under a wide variety of conditions; ionic strength was especially critical. Enterotoxin was most stable in a pH range of 5.0 to 10.0, but lost activity rapidly outside this range. Alkylation provided some protection of enterotoxin activity in crude preparations but failed to protect activity during purification procedures. It did not appear to affect critically the enterotoxin molecule itself, since elution profiles on Sephadex G-75 chromatography were unchanged after alkylation.     

Phospholipid fatty acid and lipopolysaccharide fatty acid signature lipids in methane-utilizing bacteria

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.     

Immunofluorescent Detection of Enterotoxin B in Food and a Culture Medium

Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.