Toxin In Culture Supernatants Research Articles

Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples.

A comparison of DOT-ELISA and Standard-ELISA was made for detection of Vibrio cholerae toxin in culture supernatants of bacteria isolated from human and environmental samples. A total of 293 supernatants were tested in a double blind assay. A correlation of 100% was obtained between both techniques. The cholera toxin was found in 20 Inaba and 3 Ogawa strains. Positive samples were from seafood (17 samples), potable water (1 sample) and sewage (5 samples). The DOT-ELISA was useful as the standard-ELISA to confirm the presence of cholera toxin in the environmental samples.

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

Requirement of simultaneous assessment of crystal- and supernatant-related entomotoxic activities of Bacillus thuringiensis strains for biocontrol-product development.

Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (β-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting β-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin/activity. Tests showed that 107 endospores mL−1 caused >80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted β-exotoxins, with isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems.

Detection of Clostridium difficile toxins A, B and binary toxin with slow off-rate modified aptamers

Rapid and accurate diagnostic tests for Clostridium difficile infections (CDI) are crucial for management of patients with suspected CDI and for infection control. Enzyme immunoassays for detection of the toxins are routinely used but lack adequate sensitivity. We generated slow off-rate modified aptamers (SOMAmer™ reagents) via in vitro selection (SELEX) that bind toxins A, B and binary toxin with high affinity and specificity. Using SOMAmers alone or in conjunction with antibodies, we have developed toxin assays with a 1 pmol/L (300 pg/mL) limit of detection and a 3 log dynamic range. SOMAmers proved useful as capture or detection agents in equilibrium solution binding radioassays, pull-down capture assays, dot blots, and plate- or membrane-based sandwich assays, thus represent a promising alternative to antibodies in diagnostic applications. SOMAmers detected toxins A, B and binary toxin in culture supernatants from toxigenic C. difficile, including a BI/NAP1 strain and historic strains.

Escherichia coli verotoxigênica: isolamento e prevalência em 60 propriedades de bovinos de leite da região de Pelotas, RS, Brasil

A produção de verotoxinas foi investigada em 1.127 isolamentos de Escherichia coli feitos a partir de 243 bovinos de leite, de água de consumo humano e animal e de amostras de leite de 60 propriedades da bacia leiteira de Pelotas, no período de dezembro de 1999 a dezembro de 2000, com o objetivo de determinar a prevalência de E. coli verotoxigênicas (VTEC) nas propriedades e no rebanho, de detectar a presença de sorotipos ligados a infecções humanas e de identificar, nas propriedades e na região de Pelotas, potenciais fatores de risco de infecção para os animais. A detecção das toxinas em sobrenadante de culturas de E. coli isoladas foi realizada através do ensaio de citotoxicidade em células Vero. VTEC foi isolada em 95% (57/60) das propriedades estudadas, em 49% (119/243) dos animais testados, em 5% (3/60) das amostras de água de consumo humano, em 8,35% (5/60) das amostras de água de consumo animal e em 5% (3/60) das amostras de leite. A prevalência de bovinos infectados em cada propriedade variou de 0 a 100%. Em 2,9% (7/243) dos animais testados, foram isoladas VTEC pertencentes aos sorogrupos O157, O91 e O112, que incluem cepas responsáveis por casos de colite hemorrágica e síndrome urêmica hemolítica em humanos. Fatores de risco de contaminação, como a precipitação pluviométrica, a temperatura, o tamanho da propriedade e a concentração de animais, apresentaram evidências de influenciarem a prevalência de VTEC nos animais. Estes resultados sugerem que o grupo VTEC está amplamente distribuído na bacia leiteira de Pelotas e inclui organismos pertencentes a sorogrupos patogênicos para humanos.

Shiga toxin-producing Escherichia coli (STEC) isolated from healthy dairy cattle in southern Brazil

Over a period of 1 year, the production of verotoxin was investigated in 1127 Escherichia coli isolated from 243 dairy cattle from 60 small farms in southern Brazil. Vero cell assay was used to detect toxins in culture supernatants from E. coli isolated from bovine feces. Shiga toxin-producing E. coli (STEC) detection rates were 95% (57 of 60) for farms and 49% (119 of 243) for cattle. Prevalence of STEC-positive cattle in the farms ranged from 0 to 100%. Ninety-six percent (315 of 327) of the STEC isolates did not react in the panel of sera used for typing. Twelve isolates, all non-motile, belonged to serogroups previously associated with human diseases, and 67% (8 of 12) were of only two serotypes (O91:H- and sorbitol-fermenting O157:H-). These results indicate that dairy cattle from the region surveyed may be a source of STEC potentially pathogenic for humans.

Effect of staphylococcal β toxin on the cytotoxicity, proliferation and adherence of Staphylococcus aureus to bovine mammary epithelial cells

The effect of staphylococcal β toxin on the cytotoxicity, proliferation and adherence of S. aureus to bovine mammary epithelial cells was studied. Bovine erythrocytes and mammary epithelial cells were incubated with purified staphylococcal α and β toxins and with culture supernatants from S. aureus M60 and two mutant strains that are negative for either the production of α (DU5789 α−) or β (DU5846 β−) toxin. Lysis of bovine erythrocytes was due primarily to β toxin. Alpha toxin increased the lysis of bovine erythrocytes by purified β toxin, but the presence of α toxin in culture supernatants from S. aureus did not increase the lysis of bovine erythrocytes. Purified β toxin was cytotoxic to mammary secretory epithelial cells, but to a lesser extent than α toxin. Together they exhibited an additive effect on mammary epithelial cells. Inactivation of the α toxin-gene of S. aureus M60 decreased the cytotoxic effect on mammary epithelial cells to a greater extent than the inactivation of the β toxin-gene. Also, the relative percentages of DU5789 α− and DU5846 β− adhering to mammary cell monolayers, the number and size of colonies and the number of infected epithelial cells decreased. This in vitro study showed that β toxin damages bovine mammary secretory epithelial cells, increases the damaging effects of α toxin, increases the adherence of S. aureus to mammary epithelial cells and increases the proliferation of S. aureus.

Evaluation of a reversed passive latex agglutination test for detection of Escherichia coli heat-labile toxin in culture supernatants.

One hundred strains of Escherichia coli were tested for the production of the heat-labile enterotoxin by the Y1 adrenal cell test and a commercially available reversed passive latex agglutination test. The strains were grown in Casamino Acids-yeast extract broth, and filtered culture supernatants were tested for the presence of heat-labile enterotoxin. There was perfect correlation between the Y1 test and the reversed passive latex agglutination test, and the latter was simple to perform and completed within 48 h.

Analysis of Toxic Shock Syndrome Isolates Producing Staphylococcal Enterotoxins B and Cl with Use of Southern Hybridization and Immunologic Assays

A study was undertaken to evaluate the production of enterotoxin B (Ent B), EntC1, and toxic shock syndrome toxin 1 (TSST-1) by isolates of Staphylococcus aureus from patients with toxic shock syndrome (TSS) and from a variety of other sources. Levels of toxin in culture supernatants were measured by a quantitative immunodiffusion assay. Most vaginal TSS isolates produced TSST-1, either alone or with EntC1. However, strains that produced EntB or EntC1 but did not express TSST-1 were commonly isolated from patients with nonmenstrual TSS; EntB was usually produced alone or--rarely--with EntC1. These results were confirmed by probing DNA from representative isolates with an internal probe of the EntC1 gene (entC1). Extensive sequence homology between entC1 and entB enabled detection of both genes under conditions of high stringency. The genomic location of entC1 in strains producing both EntC1 and TSST-1 varied little but was dependent on the mammalian host. In contrast, the genomic location of entC1 or entB in strains producing EntC1 or EntB alone was variable. These results suggest that these genes are contained on mobile elements.

Competitive, enzyme-linked immunosorbent assay for toxic shock syndrome toxin 1

We developed a competitive, enzyme-linked immunosorbent assay for the quantitation of toxic shock syndrome toxin 1 (TSST-1). Polyvalent immunoglobulin G from immunized rabbits was used as the capture antibody, and alkaline phosphatase conjugated to purified toxin served as the indicator enzyme. A standard curve was generated with each experiment, from which the concentration of toxin in culture supernatants was extrapolated. The assay was useful for determining toxin concentrations of 0.03 to 0.5 micrograms/ml, which is a substantial, practical improvement over immunodiffusion methods. Staphylococcal enterotoxins A through E were not significantly cross-reactive in the assay, and staphylococcal protein A did not interfere with quantitation of TSST-1. By testing a variety of staphylococcal strains, we found 100% concordance between toxin determinations made with our assay and those made by the investigators from whom the strains were obtained. The competitive, enzyme-linked immunosorbent assay is a highly reproducible, inexpensive means of determining TSST-1 concentrations and may have broad applicability in the field of toxic shock research.