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Chronic Staphylococcal enterotoxin B and lipopolysaccharide induce a bimodal pattern of hepatic dysfunction and injury.

OBJECTIVE: To determine the effect of chronic exposure to endotoxin (lipopolysaccharide) and Staphylococcal enterotoxin B on hepatic injury and function. DESIGN: Prospective, controlled trial. SETTING: Research laboratory in a university hospital. SUBJECTS: Male Sprague-Dawley rats weighing 325-350 g with chronic vascular and bile catheters. INTERVENTIONS: Chronically catheterized rats were treated daily with saline, 50 microg/kg Staphylococcal enterotoxin B alone, 1000 microg/kg lipopolysaccharide alone, 1000 microg/kg lipopolysaccharide with 50 microg/kg Staphylococcal enterotoxin B, or 100 microg/kg lipopolysaccharide with 50 microg/kg Staphylococcal enterotoxin B for 10 days. Serum and biliary measures of hepatic injury and dysfunction were measured before and then 6 hrs and 1, 2, 3, 7, and 10 days after the start of treatment. The animals were killed at 10 days and the livers examined histologically. MEASUREMENTS AND MAIN RESULTS: Mean rates of bile flow, biliary indocyanine green excretion, and bile acid flux were significantly decreased immediately after treatment (6 hr, 1 and 2 days) and then at 10 days. Increases in biliary and serum gamma-glutamyltransferase and serum bile acids also occurred in a similar bimodal pattern. Animals treated with lipopolysaccharide or Staphylococcal enterotoxin B alone became tolerant and did not develop the bimodal pattern of hepatic dysfunction. Histologic examination of the liver at 10 days revealed periportal inflammation and fibrosis. CONCLUSIONS: The combination of lipopolysaccharide and Staphylococcal enterotoxin B leads to late liver injury, whereas either toxin alone does not. These data may explain the frequent development of liver dysfunction in patients exposed to multiple bacterial toxins such as in sepsis, multiple-system organ failure, and other diseases with altered intestinal permeability.

Alanine Transaminase↗

Glucose repression of enterotoxins A, B and C and other extracellular proteins in staphlyococci in batch and continuous culture.

The production of enterotoxins, lipase and total extracellular protein by four strains of Staphylococcus aureus grown in batch culture at a controlled pH of 6.5 in a completely defined medium was markedly reduced by glucose or glycerol constantly maintained at 0.I M. A concomitant increase in the production of deoxyribonuclease, up to 13-fold, showed however that not all extracellular proteins are under the same control mechanism. The presence of glucose and glycerol in the medium also resulted in a rapid increase in the specific growth rate. However, growth of S. aureus s6 in Mgilimited continuous culture showed that glucose repression of enterotoxin B when the growth rate was held constant was more than twice that in batch culture. Therefore glucose repression can occur independently of an increase in growth rate. The specific rate of production of enterotoxin B, lipase, deoxyribonuclease, beta-haemolysin and total extracellular protein by S. aureus s6 increased as the growth rate increased from 0.07 to 0.24 h-1. Non-replicating cells grown in the absence of glucose produced considerable amounts of enterotoxin, and production was not repressed by the presence of glucose in the resuspension medium. In contrast, no enterotoxin B or C was obtained from nonreplicating cells grown in the presence of glucose. Chloramphenicol completely inhibited enterotoxin production by non-replicating cells, indicating that synthesis of new protein was required.

Bacterial Proteins↗

Assay of the heat-labile enterotoxin of Escherichia coli in infant rabbits.

Infant rabbits were shown to respond to Escherichia coli heat-labile enterotoxin by a consistent increase in intestinal fluid content, which was maximal 5 h after oral dosing. Infant rabbits could be used in a simple quantitative assay for heat-labile E. coli enterotoxin based on the ratios of gut weight to remaining body weight 5 h after oral dosing. Infant rabbits remained responsive to heat-labile enterotoxin up to 14 days of age, after which their gastric pH became low enough to destroy the enterotoxin. Rabbits that had been deprived of food before being dosed had a reduced gastric pH and a reduced response to the enterotoxin. Lincomycin andmitomycin C were found not to increase th e yield of heat-labile enterotoxin from E. coli strain P307.

Animals↗

Comparison of enterotoxins and haemolysins produced by methicillin-resistant (MRSA) and sensitive (MSSA) Staphylococcus aureus.

A collection of 201 isolates of Staphylococcus aureus was examined: 152 methicillin-sensitive S. aureus (MSSA) comprised 48 blood culture isolates (BC) and 58 isolates from routine diagnostic specimens (RD) from Glasgow Royal Infirmary (GRI), and 46 strains from nasal swabs of patients attending a general practitioner (GP); 49 isolates were of methicillin-resistant S. aureus (MRSA) from GRI. We have previously shown that the MRSA could be divided into two sub-groups on the basis of sensitivity or resistance to aminoglycoside antibiotics. Production of enterotoxins A, B, C and D, and alpha-, beta-, gamma- and delta- haemolysins was detected by reverse passive latex agglutination (RPLA) and agar overlay methods respectively: 60% of BC MSSA and a similar proportion of MSSA from other sources produced enterotoxin; 87% of aminoglycoside-sensitive MRSA produced enterotoxin (89% of these produced enterotoxin A alone) whereas only 27% of aminoglycoside-resistant MRSA were enterotoxin-positive, significantly less than either MSSA or aminoglycoside-sensitive MRSA. The proportion of haemolysin-producing isolates did not differ amongst the isolates of MSSA and MRSA; there was no difference in the distributions of haemolysins between aminoglycoside-sensitive and -resistant strains of MRSA. GP MSSA had higher and lower numbers of gamma- and delta-haemolysin producers respectively than other S. aureus isolates. alpha-Haemolysin producers were commoner amongst MRSA isolates, which were also more likely than MSSA isolates to produce several haemolysins. Differences in enterotoxin production between aminoglycoside-sensitive and -resistant MRSA isolates reflect subgroups previously defined by biotype, phage type, immunoblot and restriction enzyme fragmentation pattern data, and provide further evidence for the existence of two major MRSA clones in GRI.

Aminoglycosides↗

Improved reversed passive hemagglutination for simple and rapid detection of staphycococcal enterotoxins A approximately E in food.

Detection and identification of staphylococcal enterotoxins in food or culture filtrates were performed using the reversed passive hemagglutination (RPHA) technique, with formalized sheep red blood cells (FSRBC) sensitized with immunologlobulins of anti-A, B, C, D, and E rabbit hyperimmune sera fractionated by affinity chromatography. The FSRBC sensitized with anti-A approximately E immunoglobulins showed a high level of reactivity and specificity in RPHA, against homologous types of purified enterotoxins and culture filtrates of toxin-producing strains. No non-specific reactions with various ingredients in foods nor cross-reactions among enterotoxin types were observed. The minimum amount of enterotoxins in foods detected by RPHA was calculated to be 0.01 micorgram/g without concentration, and the recovery rate of experimentally added toxins was calculated to be about 80%. Under routine laboratory practice, detection and identification of enterotoxins from incriminated foods of five food poisoning outbreaks were performed by RPHA within 3 hr after reception of the specimens. Among them, three were determined to be enterotoxin A food poisoning, one to be toxin C and the rest to be intoxication of A and D. The concentrations of the toxins was between 0.014 and 3.65 microgram per gram of food.

Disease Outbreaks↗

Prevention of lung metastasis by intra-tumoral injection of Cepharanthin and staphylococcal enterotoxin B in transplantable rat osteosarcoma.

The antitumor effect of intra-tumoral injection of Cepharanthin, a biscoclaurin alkaloid extracted from Stephania cephalanta Hayata, and staphylococcal enterotoxin B was evaluated using F344 male rats bearing transplantable rat osteosarcoma, S-SLM. A macroscopic lung metastatic nodule of tumor was transplanted into the subcutaneous back space, and 0.5 mg of Cepharanthin and 2 pg of staphylococcal enterotoxin B were injected into the tumor on days 12, 13 and 14. On day 28, all animals were killed with an overdose of pentobarbital sodium, and the transplanted tumors and lungs were examined. The wet weight of the lungs of the rats treated with Cepharanthin and staphylococcal enterotoxin B was significantly lower, and apoptosis in the lung metastatic nodules was significantly higher than that of the control or that of rats treated with only Cepharanthin or staphylococcal enterotoxin B. In the transplanted tumors, infiltration of TRAP (tartrate-resistant acid phosphatase)-positive multinucleated giant cells was prominent in the rats treated with Cepharanthin and staphylococcal enterotoxin B. These findings indicate that intra-tumoral injection of Cepharanthin and staphylococcal enterotoxin B induced infiltration of TRAP-positive multinucleated giant cells within the transplanted rat osteosarcoma, and reduced lung metastasis.

Acid Phosphatase↗

Isolation, primary structure and synthesis of heat-stable enterotoxin produced by Yersinia enterocolitica.

Five heat-stable enterotoxins were isolated from the culture supernatant of Yersinia enterocolitica and purified to homogeneity by DEAE-Sephacel and high-performance liquid chromatographies. They caused acute fluid accumulation in the intestine of suckling mice. The amino acid sequence of one of the enterotoxins was determined to be Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys, by Edman degradation of its pyridylethylated derivative and a combination of fast atom bombardment mass spectrometry and carboxypeptidase B digestion. This structure was unambiguously confirmed by chemical synthesis. The other enterotoxins had longer or shorter amino acid sequences at their N termini, but the same sequence at their C termini. The six half-cystine residues formed intramolecular disulfide linkages, as shown by measurement of the molecular masses of the enterotoxins by fast atom bombardment mass spectrometry. The sequence of 13 amino acid residues at the C terminus showed similarity to those of heat-stable enterotoxins isolated from enterotoxigenic Escherichia coli [Aimoto, S. et al. (1982) Eur. J. Biochem. 129, 257-263; Takao, T. et al. (1983) FEBS Lett. 152, 1-5] suggesting that these similar sequences are related to the common biological and immunological properties of enterotoxins produced by Y. enterocolitica and enterotoxigenic E. coli.

Amino Acid Sequence↗

Chemical modification and characterization of enterotoxin from clostridium perfringens type A.

Enterotoxin from Clostridium perfringens type A has been purified. The enterotoxin was shown to be heat-labile, but re-activation of heat treated enterotoxin did occur down to an activity of 15 per cent of the native enterotoxin. The molecular weight was shown to be 34,000 by ultracentrifugation, and the molecular weight did not change significantly after treatment with 0.1 M beta-mercaptoethanol and 6 M guanidine hydrochloride. It was concluded that the enterotoxin consists of one single polypeptide chain. The enterotoxin did not lose any activity after treatment with iodoacetic acid and iodoacetamide, but a complete loss of activity was observed after succinylation.

Amino Acids↗

The effect of Ca++ and Mg++ on the action of Clostridium perfringens enterotoxin on Vero cells.

Clostridium perfringens enterotoxin binds to receptors on Vero cells. This process does not depend on the presence of divalent cations (Ca++, Mg++). Binding of enterotoxin causes inhibition of 14C-leucine incorporation into proteins, probably because of depression of amino acid transport. The presence of Mg++ speeds up this effect of the enterotoxin. The enterotoxin produces membrane leakage only in the presence of Ca++, but additional Mg++ increases the rate of this process. These results indicate that the dissociation constant of the enterotoxin receptor interaction is reduced in the presence of Mg++. A model for the mode of action of the enterotoxin is proposed.

Amino Acids↗

Raffinose increases sporulation and enterotoxin production by Clostridium perfringens type A.

Replacement of starch with raffinose in Duncan and Strong sporulation medium improved percent sporulation in six of eight strains tested. Enterotoxin concentration in cell extracts was increased in the case of four of five known enterotoxin-positive strains. With strain NCTC 10240, levels of 0.3, 0.4, and 0.5% raffinose produced the highest enterotoxin concentration 300 to 320 micrograms of enterotoxin per mg of cell extract protein. At a level of 0.4% raffinose the highest enterotoxin concentration in cell extracts of NCTC 10240 occurred after 8 h of growth in Duncan and Strong medium. Enterotoxin produced in the presence of starch or raffinose by three separate strains all migrated at similar Rm by polyacrylamide gel electrophoresis.

Clostridium perfringens↗

Determination of staphylococcal enterotoxin A in cheddar cheese produced without starter activity.

Three variants of the chloramine-T radioiodination method were used to iodinate staphylococcal enterotoxin A with 125I. Only one method consistently produced usable labels for radioimmunoassay. The iodine incorporation was 55 to 76%; the specific activity was 3.5 to 5.5 muCi/microgram of enterotoxin, and the label was extremely stable on storage at -20 degrees C. Determinations of the enterotoxin in extracts of cheddar cheese produced without starter activity were carried out with the radioimmunoassay system and protein A as antibody immunoadsorbent. The assay buffer used in this system significantly influenced the detected levels of enterotoxin in the cheese extracts. Phosphate buffer, but not tris(hydroxymethyl)aminomethane (Tris) buffer, caused gelling of cheese extract proteins, thus resulting in an incomplete separation of free from antibody-bound 125I enterotoxin. When Tris buffer was used, the results indicated a high degree of accuracy and precision for this radioimmunoassay. The lowest detectable enterotoxin concentration in cheese extract was 0.5 ng/ml.

Animals↗

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.

Animals↗

Production of staphylococcal enterotoxins C1 and C2 and thermonuclease throughout the growth cycle.

Synthesis of enterotoxins C1 and C2 and thermonuclease throughout the growth cycle was investigated with Staphylococcus aureus type strains FRI137 and FRI361 and S. aureus isolates M5 (C1) and L2 (C2) of animal origin. Both enterotoxins were produced during the exponential growth phase or at the beginning of the stationary phase. The minimal incubation time (7 to 12 h) and the lowest population (10(7) to 2 x 10(9) CFU/ml) associated with detectable enterotoxin (1 to 6.5 ng/ml) were related to the total amount of toxin produced after 24 h. Thermonuclease was detected in all samples whenever enterotoxins were detected. Furthermore, strain FRI137 produced thermonuclease earlier and at lower cell populations than it did enterotoxin C1. Patterns of enterotoxin and thermonuclease synthesis did not correlate. The concentration of toxins increased throughout the growth cycle, while the concentration of thermonuclease remained constant during the last hours of the growth cycle.

Animals↗

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.

Enterotoxins↗

Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit (TECRA).

The TECRA kit, a commercial staphylococcal enterotoxin visual immunoassay kit, is an enzyme-linked immunosorbent assay system which utilizes polyvalent antisera against staphylococcal enterotoxin types A to E. The test is simple and rapid to perform (4 h) and has therefore been widely used for screening purposes. In this study, the TECRA kit produced a number of false-positive reactions with seafood; 25% of 218 samples of seven types of seafood gave false-positive results, particularly shellfish such as mussels (85%), clams (32%), oysters (23%), winkles (20%), and squid (13%). Some nonshellfish samples also gave false-positive results with the TECRA kit (smelt [20%] and trout [10%]). The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was: (i) heat labile, being completely inactivated by heating for 3 min at 70 degrees C, compared with 5% inactivation of true staphylococcal enterotoxins by the same heat treatment, (ii) in a selective reaction with normal rabbit or calf serum (nonspecific reactions were completely abolished by these sera, whereas staphylococcal enterotoxins were not affected), and (iii) incapable of binding to a copper-chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The false-positive reactions occurring with seafood were not associated with substances produced by microorganisms, since the bacterial isolates from the samples did not give positive results with the TECRA kit.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections.

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.

Animals↗

Production of staphylococcal enterotoxins A, B, and C in various media.

The effect of initial pH and of length of incubation time at 37 C in four different growth media on the production of staphylococcal enterotoxins A, B, and C was determined. A starting pH of 6.8 gave higher yields of enterotoxins B and C than either pH 6.0 or 5.3. The production of enterotoxin A was, however, not materially affected by the low initial pH of 5.3. Prolonged incubation (48 to 72 hr) resulted only occasionally in higher yields of enterotoxin. The effect of the media on the amount of enterotoxin produced is considerable. Difco Brain Heart Infusion (BHI) was inferior to either Fisher BHI, 4% NZ Amine (NAK), or 3% NAK plus 3% protein hydrolysate powder at the three initial pH values, regardless of length of incubation time. The slight effect of the low starting pH on the production of enterotoxin A is being further investigated.

Culture Media↗

Factors affecting the secretion of staphylococcal enterotoxin A.

The biosynthesis of enterotoxin A by replicating and nonreplicating cells was investigated. Unlike enterotoxin B, a secondary metabolite, enterotoxin A secretion resembled that of a primary metabolite by being secreted during the exponential phase of growth. The amount of toxin produced per unit of growth was not influenced by NaCl, NaNO(2), or NaNO(3). Several surfactants increased toxin secretion. Toxin secretion by nonreplicating cells was inhibited by chloramphenicol and 2, 4-dinitrophenol but not by streptomycin or penicillin G. The optimal pH for enterotoxin A production was 6.5 to 7.0. The findings suggest a number of possible reasons for the higher incidence of food poisonings caused by enterotoxin A as compared to enterotoxin B.

Chloramphenicol↗