Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection.

Two phosphonodehydrotripeptides: Boc0-Gly1-Δ(Z)Phe2-α-Abu3PO3Me2 and Boc0-Gly1-Δ(Z)Phe2-α-Nva3PO3Et2.

The present paper reports the crystal structures of two short phosphonotripeptides (one in two crystal forms) containing one ΔPhe (dehydrophenylalanine) residue, namely dimethyl (3-{[tert-butoxycarbonylglycyl-α,ß-(Z)-dehydrophenylalanyl]amino}propyl)phosphonate, Boc(0)-Gly(1)-Δ(Z)Phe(2)-α-Abu(3)PO3Me2, C21H32N3O7P, (I), and diethyl (4-{[tert-butoxycarbonylglycyl-α,ß-(Z)-dehydrophenylalanyl]amino}butyl)phosphonate, Boc(0)-Gly(1)-Δ(Z)Phe(2)-α-Nva(3)PO3Et2, as the propan-2-ol monosolvate 0.122-hydrate, C24H38N3O7P·C3H8O·0.122H2O, (II), and the ethanol monosolvate 0.076-hydrate, C24H38N3O7P·C2H6O·0.076H2O, (III). The crystals of (II) and (III) are isomorphous but differ in the type of solvent. The phosphono group is linked directly to the last Cα atom in the main chain for all three peptides. All the amino acids are trans linked in the main chains. The crystal structures exhibit no intramolecular hydrogen bonds and are stabilized by intermolecular hydrogen bonds only.

Production of staphylococcal enterotoxin R by Staphylococcus aureus strains.

Staphylococcal enterotoxin D and R (SED, SER) production was determined in 24 S. aureus strains harboring sed gene. Seven of them were not able to produce SED as evidenced by enzyme-linked immunosorbent assay and Western blotting. Sequencing revealed that all these strains harbor a variant of sed gene. Expression of SER was detectable in 22 out of 24 isolates, with variance in productivity ranging from ∼40 to 450 ng/mL. Out of the seven isolates not able to produce SED, three produced high amounts of SER (249-396 ng/mL), two produced less than 200 ng/mL of SER, and two were found to express no detectable amount of SER. Three of those were assigned to spa type t1677 with two being of agr type III and one of agr type I. One strain was t084, agr type II, one t603, agr type II, one 2920, agr type III, one t2920, agr type III, and one t5160, agr type I. Because conventional screening procedures involve only the detection of classical enterotoxins in food, the isolates not able to produce SED presented in this study could pose a threat to human health due to SER production.

Temporal expression of staphylococcal enterotoxin h in comparison with accessory gene regulator-dependent and -independent enterotoxins.

Using sandwich enzyme-linked immunosorbent assay (ELISA), the production of staphylococcal enterotoxin (SE) H was determined in 22 Staphylococcus aureus isolates bearing the seh gene. Samples of supernatants were taken at four time points corresponding to exponential phase (optical density at 600 nm [OD(600)] 0.3 to 0.6), late exponential phase (OD(600) 2 to 4), early stationary phase (OD(600) 4 to 6), and late stationary phase (OD(600) 7 to 12). In four isolates, SEH was detectable at a very low level at the first time point. In 18 isolates, the earliest SEH production was detected in the late exponential phase. For all isolates, there was an increase of SEH concentration with time. Western blot analysis revealed that SEH production, similar to SEA, started in the early exponential phase (OD(600) ∼ 0.5). Isolates with high SEH productivity, as measured by ELISA, demonstrated a higher seh transcription as well. sec transcription was induced in the stationary phase. An induction in the sea transcript was observed during mid- to late exponential phase. Expression profile of seh was similar to that of sea. We showed that the seh expression profile is similar to that of Agr-independent sea and not to that of Agr-dependent sec genes. SEH can be effectively expressed at low bacterial counts, meaning that even in an environment not favorable for S. aureus growth, seh-bearing strains can pose a risk for food safety.

Characterization of borderline oxacillin-resistant Staphylococcus aureus isolated from food of animal origin.

In this study, the molecular characteristics of food-derived oxacillin-resistant Staphylococcus aureus were determined. Eight borderline oxacillin-resistant strains with MICs of 2 to 4 microg/ml were identified from 132 S. aureus isolates of food origin. One of the two isolates with a MIC of 4 microg/ml was methicillin-resistant determinant (mecA) gene positive, and the other six with MICs of 2 microg/ml were mecA negative. The mecA-positive isolate was classified as sequence type (ST)228, staphylococcal protein A (spa) type t041, and carried the staphylococcal cassette chromosome mec type I element. Two borderline oxacillin-resistant strains were classified as spa t008 and ST8, and the remaining five as spa t164 and ST20. The mecA-positive strain and four borderline oxacillin-resistant strains were found enterotoxigenic. The enterotoxin genes detected in these strains included selp, egc1, and sed-sej-selr. The borderline-resistant S. aureus isolates from a manually handled product, i.e., minced pork, were shown genetically related to strains associated with human infections. This suggests that humans can be considered as a source of contamination of this food with oxacillin-resistant S. aureus strains. The genotypes of the investigated milk borderline-resistant isolates were shown to occur not only in cows, but also in humans. Since manual handling is reduced in raw milk production, a human origin of S. aureus seems unlikely. Because knowledge of the genotypes of animal staphylococci is limited, more research is needed to address the question of the origin of antibiotic-resistant S. aureus strains in food.

Enterotoxin gene content in Staphylococcus aureus from the human intestinal tract.

Enterotoxigenic Staphylococcus aureus has been associated with staphylococcal food poisoning, which in a number of patients is accompanied by gastroenteritis. It has also been found to persist asymptomatically in the human intestinal tract, being considered one of the sources of pathogen transmission to manually handled food. However, very little is known about the incidence and enterotoxigenicity of intestinal S. aureus not associated with enteritis. There are practically no data on the frequency of some enterotoxin genes in intestinal S. aureus. Six thousand six hundred and twenty-one fecal swabs from 6-month- to 8-year-old children were analyzed for S. aureus. Growth of S. aureus was found in 347 samples. Two hundred and eight S. aureus isolates (4.2% of 4900 swabs) were from patients with sporadic enteritis and 139 isolates (8% of 1721 swabs) from patients who did not develop diarrhea during hospitalization. The genes encoding 16 members of the enterotoxin family (sea-see, seg-selp, and selu) and tst were present in 174 (83%) S. aureus isolates accompanying enteritis and in 101 (72%) isolates derived from patients with no enteritis symptoms. The genes of the classical enterotoxins (sea-see) and tst were present in 56% and 59% of the enteritis-associated and nonenteritic isolates, respectively.