Quantitative Analysis of Nitrofuran Metabolites and Chloramphenicol in Shrimp Using Acetonitrile Extraction and Liquid Chromatograph-Tandem Mass Spectrometric Detection: A Single Laboratory Validation.

A method was developed and validated for the simultaneous analysis of chloramphenicol and nitrofuran metabolites in shrimp according to the guideline established by the U.S. Food and Drug Administration Office of Foods and Veterinary Medicine. The extraction steps following the overnight hydrolysis and derivatization are simpler than the conventional ethyl acetate extraction method. The main steps are neutralization of hydrolysates, addition of acetonitrile for extraction, and salting out of organic phase from the acetonitrile-aqueous mixture. Extracts are analyzed for chloramphenicol and nitrofuran metabolites by LC-MS/MS in a single injection with polarity switching between the positive electrospray ionization (ESI) mode for the nitrofurans and the negative ESI mode for chloramphenicol. Recoveries calculated using an extracted matrix calibration curve and labeled internal standards for chloramphenicol and nitrofurans ranged from 98.6 to 109.2% with RSDs less than 18%. This method that combines the analysis of chloramphenicol with the nitrofurans was shown to generate analytical results similar to those obtained using the individual drug-class analytical methods currently used for the analysis of chloramphenicol or nitrofurans in shrimp.

Determination of total nitrofuran metabolites in shrimp muscle using liquid chromatography/tandem mass spectrometry in the atmospheric pressure chemical ionization mode.

The method of MacMahon and Lohne for analysis of nitrofuran metabolites in shrimp was optimized to streamline the extraction processes and the LC analysis. This revised method includes 16 h of mild acid hydrolysis/derivatization followed by ethyl acetate extraction and analysis by LC/MS/MS in the atmospheric pressure chemical ionization mode. This revised method was validated in shrimp for concentrations of 0.25 to 2.0 ng/g. The LOQ was 0.25 ng/g for all metabolites. The LOD was 0.052 nglg for 1-aminohydantoin (AHD), 0.206 ng/g for 3-amino-2-oxazolidinone (AOZ), 0.108 ng/g for semicarbazide (SC), and 0.062 ng/g for 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The spike recoveries with RSD into negative matrix at 1 ng/g were 100.2% (3.2%) for AHD, 102.5% (1.0%) for AOZ, 103.7% (2.3%) for SC, and 104.0% (3.3%) for AMOZ. The spike recoveries at 1 ng/g into unknown samples (n=108) containing varied levels of nitrofuran metabolites were 112.6% (25.7%) for AHD, 108.1% (12.1%) for AOZ, 103.0% (12.0%) for SC, and 100.3% (6.9%) for AMOZ. Interday precision with samples containing incurred AOZ concentrations of 0.92 to 17.8 ppb performed over a year was 10.4% RSD. The method is accurate and precise for determining nitrofuran concentrations in the edible tissue of shrimp.

Multidrug-resistant Klebsiella pneumoniae isolated from farm environments and retail products in Oklahoma.

Multidrug-resistant enteric bacteria were isolated from turkey, cattle, and chicken farms and retail meat products in Oklahoma. Among the isolated species, multidrug-resistant Klebsiella pneumoniae was prevalently isolated from most of the collected samples. Therefore, a total of 132 isolates of K. pneumoniae were characterized to understand their potential roles in the dissemination of antibiotic-resistance genes in the food chains. Multidrug-resistant K. pneumoniae was most frequently recovered from a turkey farm and ground turkey products among the tested samples. All isolates were resistant to ampicillin, tetracycline, streptomycin, gentamycin, and kanamycin. Class 1 integrons located in plasmids were identified as a common carrier of the aadA1 gene, encoding resistance to streptomycin and spectinomycin. Production of beta-lactamase in the K. pneumoniae isolates played a major role in the resistance to beta-lactam agents. Most isolates (96%) possessed bla(SHV1). Five strains were able to express both SHV-11 (pI 6.2) and TEM-1 (pI 5.2) beta-lactamase. Transfer of these antibiotic-resistance genes to Escherichia coli was demonstrated by transconjugation. The bacterial genomic DNA restriction patterns by pulsed-field gel electrophoresis showed that the same clones of multidrug-resistant K. pneumoniae remained in feathers, feed, feces, and drinking water in turkey environments, indicating the possible dissemination of antibiotic-resistance genes in the ecosystem and cross-contamination of antibiotic-resistant bacteria during processing and distribution of products.

Development of immunoassay for detection of meat and bone meal in animal feed.

An immunoassay system was developed for efficient detection of prohibited meat and bone meal (MBM) in animal feed. Monoclonal antibodies (MAbs) were raised against bovine smooth muscle autoclaved at 130 degrees C for 20 min. Among the 1,500 supernatants of hybridoma cells screened, MAbs 3E1, 1G3, and 3E10 were selected and characterized in this study. The first set of MAbs produced, 3E1 and 1G3, had stronger reactivity against MBM than against smooth muscle that was heat treated at 90 degrees C for 10 min. However, reactivity gradually increased against smooth muscle that was autoclaved at 130 degrees C for up to 1 h. The enzyme-linked immunosorbent assay for detection of MBM in animal feed was optimized with the MAb 3E10 because of its superior performance. MAb 3E10 diluted to 100-fold was used to differentiate bovine MBM from that of other species in ingredients used for commercial animal feeds and could detect down to 0.05% MBM mixed in animal feed.

Molecular characterization of multidrug-resistant Proteus mirabilis isolates from retail meat products.

Sixty-four multidrug-resistant isolates of Proteus mirabilis were collected from retail meat products in Oklahoma. The isolates showed four different patterns of antibiotic resistance based on their resistant phenotype and genotypes. Most of these isolates were resistant to ampicillin, tetracycline, gentamycin, and kanamycin. Class 1 integrons were detected as a common carrier of the antibiotic-resistant genes, such as aadA1, aadB, and aadA2. A few isolates (9%) contained class 2 integrons with three gene cassettes included: dhfr1, sat1, and aadA1. These isolates were even resistant to nalidixic acid due to mutations in gyrA and parC. All ampicillin-resistant isolates contained blaTEM-1. Plasmids that contained class 1 or 2 integrons and blaTEM-1 were able to be transferred from P. mirabilis isolates into Escherichia coli by conjugation, indicating that conjugal transfer could contribute to the dissemination of antibiotic resistance genes between the Enterobacteriaceae species.

Kenojeinin I, antimicrobial peptide isolated from the skin of the fermented skate, Raja kenojei.

An antimicrobial peptide was purified from fermented skate skin extract using the solid-phase extraction and separation on HPLC reversed-phase chromatography. Amino acid sequence of the purified peptide (Peak A) having an antimicrobial activity revealed the presence of many cationic residues of the total 28 amino acids. Its molecular mass was found to be 3059 Da. This result was in excellent agreement with the theoretical molecular mass calculated from the amino acid sequence. The synthetic kenojeinin I had inhibitory effects on B. subtilis (MIC, 12 microg/ml), E. coli (28 microg/ml), and S. cerevisiae (12 microg/ml). These results indicate that fermented skate skin is potentially antimicrobial.

Purification and determination of inhibitory activity of recombinant soyacystatin for surimi application.

A recombinant soyacystatin (r-soyacystatin) was tested for its inhibitory activity against cysteine proteinase of Pacific whiting and its activity was compared to that of egg white cystatin. A recombinant soyacystatin expressed in Escherichia coli was purified to electrophoretic homogeneity using phenyl-Sepharose and DEAE-Sepharose. Native egg white cystatin was purified by using affinity chromatography on CM-papain-Sepharose generated in our lab. Egg white cystatin and soyacystatin were tested for proteinase inhibitory activity against commercial papain and also cathepsin L purified from Pacific whiting muscle. The r-soyacystatin exhibited papain-like protease inhibition activity comparable to that of the egg white cystatin, which could inhibit papain and Pacific whiting cathepsin L. The r-soyacystatin subsequently inhibited the autolytic activity of Pacific whiting surimi.

Production of monoclonal antibody for the detection of meat and bone meal in animal feed.

For the detection of prohibited meat and bone meal (MBM) in animal feed, monoclonal antibodies (MAbs) were raised against heat-stable h-caldesmon purified from bovine intestinal smooth muscle. The obtained hybridoma cells were screened against extracts of the bovine MBM and heat-treated smooth muscle, and MAb 5E12 was identified as having the best performance. Antibody 5E12 did not react with animal feed, milk product, plant proteins, and other ingredients used for commercial animal feed except for the gelatin. This antibody diluted to 100-fold was able to detect MBM mixed in animal feed at 0.05% in an ELISA, and it showed strong affinity toward bovine smooth muscle autoclaved at 130 degrees C. Therefore, this antibody can be used in the ELISA system for field testing of the presence of MBM in animal feed.

Detection of Morganella morganii, a prolific histamine former, by the polymerase chain reaction assay with 16S rDNA-targeted primers.

A polymerase chain reaction (PCR) assay for the rapid and sensitive detection of the most prolific histamine former, Morganella morganii, was developed. 16S rDNA targeted PCR primers were designed, and the primer specificity and sensitivity of the PCR assay were evaluated. The 16S rDNA sequence (1,503 bp) for M. morganii showed 95% identity to those for enteric bacteria, i.e., Enterobacter spp., Klebsiella spp., Citrobacter spp., Hafnia alvei, Proteus spp., and Providencia spp. The unique primers for M. morganii were designed on the basis of the variable regions in the 16S rDNA sequence. The primers showed positive reactions with all M. morganii strains tested. However, PCR amplification was not detected when the primers were tested with other enteric or marine bacteria. When the sensitivity of the assay was evaluated, M. morganii was detected at levels ranging from 10(6) to 10(8) CFU/ml in albacore homogenate after the PCR amplification. The sensitivity of the assay was greatly improved with the enrichment of samples, and 9 CFU of M. morganii per ml of albacore homogenate was detected after 6 h of enrichment at 37 degrees C.

Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle.

A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.