Binding of collagen I to Escherichia coli O157:H7 and inhibition by carrageenans.

Research at USDA attempts to eliminate or reduce Escherichia coli contamination in meat and poultry foods by understanding the attachment mechanisms. This study utilizes a surface plasmon resonance (SPR) biosensor to determine the interactions of the immobilized E. coli O157:H7 surface with collagen I and selected polysaccharides. The binding and dissociation kinetics of collagen I with E. coli surface molecules had a mean affinity constant (K) of 3 x l0(8) (M(-1)) while the dissociation rate was 4.4 x l0(-5) (S(-1)). Using the SPR biosensor, carrageenan, sodium alginate and pectin were evaluated for their interactions with collagen I and the E. coli surface. Results showed 89% to 100% inhibition by carrageenans and about 50% by sodium alginate and less than 10% by pectin. The biosensor binding studies were augmented by the scanning electron microscopy studies, which also showed the attachment of E. coli to the collagen fibrils of the bovine tissues. These studies serve as the basis for developing new strategies to block bacterial attachment or detach pathogens from animal carcasses.

Identification of beta-lactam antibiotics in tissue samples containing unknown microbial inhibitors.

Antibiotic residues in animal tissues can be detected by various screening tests based on microbial inhibition. In the 7-plate assay used by the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS), penicillinase is incorporated into all but one plate to distinguish beta-lactam antibiotics from other types. However, beta-lactams such as cloxacillin and the cephalosporins are resistant to degradation by penicillinase. They may not be identified as beta-lactams by this procedure, and thus, they may be identified as unidentified microbial inhibitors (UMIs). However, these penicillinase-resistant compounds can be degraded by other beta-lactamases. The present study describes an improved screening protocol to identify beta-lactam antibiotics classified as UMIs. A multiresidue liquid chromatographic procedure based on a method for determining beta-lactams in milk was also used to identify and quantitate residues. The 2 methods were tested with 24 tissue FSIS samples classified as containing UMIs. Of these, 3 contained penicillin G, including one at a violative level, and 5 contained a metabolite of ceftiofur. The others were negative for beta-lactam antibiotics.

A screening method for beta-lactams in tissues hydrolyzed with penicillinase I and lactamase II.

Antibiotic residues above tolerance levels are not allowed in foods derived from farm animals. Microbial inhibition assays are used to screen antibiotics in U.S. regulatory laboratories. We developed a screening approach to classify beta-lactams through selective hydrolysis of the beta-lactam ring with Penase or lactamase II, thereby inactivating the beta-lactam activity. Optimum conditions for hydrolysis of beta-lactams with Penase and lactamase II were determined. beta-Lactams were detected by a microbial inhibition assay and with enzyme-linked immunosorbent assays before and after hydrolysis. beta-Lactams (10-100 ppb) were spiked in kidney extracts and hydrolyzed. Results indicate a pattern that tentatively classified the beta-lactams into 3 subgroups. Desfuroyl-ceftiofur-cysteine, a major metabolite of ceftiofur, was clearly detected. Penicillin G, ampicillin, amoxicillin, and cloxacillin were distinguishable from cephapirin, ceftiofur metabolite, and high levels of hetacillin. Liver and kidney tissue samples were analyzed with the combined enzyme hydrolysis and screening assays, which tentatively identified the residues. This approach can speed up screening analysis of beta-lactam residues prior to identification and quantitation by chromatographic analysis, thus enhancing positive identification of residues to provide a safer food supply.

Development of a chromatographic method for the isolation and detection of hygromycin B in biological fluids.

An affinity chromatography method was developed for the purification of hygromycin B from biological fluids. Lysozyme and alpha-lactalbumin were immobilized on an N-hydroxysuccinimide activated agarose support. Hygromycin B solubilized in water was bound by the proteins and subsequently eluted using 10 mM sodium citrate buffer, pH 4.0. Hygromycin B was purified from swine plasma, bovine serum and bovine milk samples using a combination of ion-exchange chromatography for initial clean-up of spiked biological samples followed by affinity chromatography. Thin layer chromatographic analysis of the isolated hygromycin B revealed one band with the same R(F) value as the hygromycin B standard.

Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides.

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective RF values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.

Improved thin-layer chromatographic detection of diethylstilbestrol and zeranol in plasma and tissues isolated with alumina and ion-exchange membrane columns in tandem.

Clean-up procedures for the isolation of zeranol and diethylstilbestrol (DES) were modified to reduce the analysis time and to increase the efficiency of purification. Several dyes (Fast Blue BB, Fast Corinth V, Fast Blue RR, Fast Blue B, Fast Red Violet B and Fast Violet B) were evaluated, and their minimum detectabilities were determined. Conditions for non-instrumental, semi-quantitative thin-layer chromatography were optimized. Zeranol and DES in plasma and tissues were determined using modified procedures. Enzyme digestion brought about significant improvement in detectabilities of zeranol and DES in both fortified and incurred plasma, serum and tissues. Minimum detectabilities for zeranol and DES were 25 ppb in fortified plasma and tissues. The amount of incurred zeranol measured in the serum of an experimental cow was increased four times, i.e. from 50 to 200 ppb, after protease digestion. Glucuronidase digestion showed an eight-fold increase in detection of incurred zeranol levels in bovine liver eight times. These results suggest that digestion releases zeranol and DES from protein and glucuronide complexes, thereby allowing detection of low levels of zeranol and DES which may not be detectable without digestion. Further modification of the purification with an ion-exchange membrane reduced the analysis time by 25%, and the membranes were regenerated up to ten times without loss of activity, allowing an automated process. This method utilizes inexpensive equipment and avoids use of organic solvent, in this case diethyl ether.

Thin-layer chromatographic detection of zeranol and estradiol in fortified plasma and tissue extracts with Fast Corinth V.

In an attempt to improve sensitivity of thin-layer chromatographic (TLC) analysis and selectivity of visualizing agents for detection of estrogenic anabolic hormones, several dyes were screened for their chromogenic interactions with estrone, estradiol, diethylstilbestrol (DES), zeranol (zearalanol), zearalanone, and mycotoxins, zearalenone and zearalenol. Fast Corinth V salt was selected for its relatively high sensitivity. These anabolic compounds were separated by TLC and visualized with Corinth V and the results compared to iodine and starch visualization. Fortified bovine plasma and tissues (kidney, liver and muscle) and chicken muscles were analyzed after a clean-up procedure using solid-phase dual columns of alumina and anion-exchange resin. Iodine-starch clearly detected 4 ng of estradiol and DES while zeranol and zearalenone were detected at higher levels (10 ng). Fast Corinth V showed distinct spots with 2 ng of zeranol and 4 ng of zearalenone while faint spots were observed with estradiol and estrone standards. DES was not detectable at these levels. Less background interference was observed with Corinth V than with iodine-starch. The former confirmed spots detected by iodine-starch. This study suggests its selectivity for detection of zeranol and its metabolite, zearalanone, in the presence of steroidal compounds.

A multi-residue TLC screening procedure for anabolic oestrogens and detection of oestradiol, DES or zeranol in chicken muscle tissue extracts.

A multi-residue HETLC (High Efficiency Thin Layer Chromatography) screening procedure for 17 beta-oestradiol, diethylstilboesterol (DES), zearalanol (zeranol), zearalenone and their metabolites oestrone, zearalanone, and zearalenol is described. The anabolic oestrogens were analyzed on HETLC plates coated with silica gel and were developed in methylene chloride:methanol: 2-propanol (97:1:2 v/v). The spots were visualized by exposure to iodine vapours and subsequently sprayed with 1% starch solution. Analysis of standards by HETLC at 4 degrees C as a seven-component mixture showed six discrete bands with mean Rfs of 0.37 (oestrone), 0.35 (zearalanone and zearalenone), 0.26 (t-DES), 0.23 (oestradiol), 0.17 (zearalenol and zearalanol), and 0.15 (c-DES). Chicken muscle tissues (1, 2.5, or 5 g) were extracted with 95% acetone. Extracts were then fortified with 50-250 ng each of the anabolic oestrogens, purified in alumina and ion-exchange columns and analyzed by HETLC. Oestradiol, zeranol or DES in fortified tissue extracts were clearly detected when an equivalent of 4 ng were analyzed by HETLC after purification in alumina and ion-exchange columns. The intensity of their bands suggested near quantitative recovery when compared to intensity of bands of known amounts of standards. The described extraction, purification, and TLC procedures can be used to screen these oestrogens at low ppb amounts in chicken muscle tissues and should be applicable to screen tissues of cattle and sheep.

High performance liquid chromatographic separation of anabolic oestrogens and ultraviolet detection of 17 beta-oestradiol, zeranol, diethylstilboestrol or zearalenone in avian muscle tissue extracts.

A multi-residue analysis was developed to discern oestrogens (17 beta-oestradiol or zeranol) used as anabolic drugs and diethylstilboestrol (DES) from the mycotoxins zearalenone and zearalenol. A mixture of 17 beta-oestradiol, oestrone, DES, zeranol, zearalenol, zearalenone and zearalanone was analysed using a 5 micron silica column with gradient elution from hexane to hexane:methanol:2-propanol, 40:45:15 (v/v) and ultraviolet detection at 280 nm. Only six peaks were obtained since zearalenone and zearalanone had identical elution times. Minimum detectabilities were 5-10 ng for the standards. Chicken muscle tissues (2.5 g) were extracted with acetone:water (95:5, v/v). The extracts were spiked with oestradiol and zeranol at 50, 100 or 200 ppb; and DES or zearalenone at 50 ppb; cleaned-up through dual columns of basic alumina and phosphate exchanged AGMP-1 resin and analysed by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Minimum detectability was 10 ng for oestradiol, zeranol, DES or zearalenone when tissues were spiked at 50 ppb. Recoveries were greater than or equal to 86%, greater than or equal to 83%, 60% and 100% for oestradiol, zeranol, DES and zearalenone, respectively.