Sue A. Reed

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.

Capture of Escherichia coli O157:H7 Using Immunomagnetic Beads of Different Size and Antibody Conjugating Chemistry.

Immunomagnetic beads (IMB) were synthesized using anti-Escherichia coli O157 antibodies and magnetic beads of two different sizes (1 μm and 2.6 to 2.8 μm) that contained a streptavidin coating, activated carboxyl groups or tosylated surfaces. The synthesized IMB, together with a commercially available IMB, were used to capture different strains of E. coli O157:H7 and E. coli O157:NM. The E. coli capture was measured by the time resolved fluorescence (TRF) intensity using a sandwich assay which we have previously demonstrated of having a sensitivity of 1 CFU/g after 4.5 hour enrichment [1]. The analyses of measured TRF intensity and determined antibody surface concentration indicated that larger beads provided higher response signals than smaller beads and were more effective in capturing the target of interest in pure culture and ground beef. In addition, while each type of IMB showed different favorable capture of E. coli O157:H7, streptavidin-coated IMB elicited the highest response, on average. Streptavidin-coated IMB also provided an economic benefit, costing less than

Complete Genome Sequence of Campylobacter jejuni YH001 from Beef Liver, Which Contains a Novel Plasmid.

0.50 per assay. The results could be used to guide the proper choice of IMB for applications in developing detection processes for E. coli O157:H7.

Whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken reveals a plasmid-borne type VI secretion system

ABSTRACT Campylobacter jejuni, commonly found in poultry and meat products, causes gastroenteritis in humans. Here, we report the complete genome sequence of a C. jejuni strain, YH001, isolated from retail beef liver. The genome is 1,712,361 bp and has a 30.5% G+C content and two plasmids of 46.5 kb and 4.4 kb.

Apparent Thixotropic Properties of Saline/Glycerol Drops with Biotinylated Antibodies on Streptavidin-Coated Glass Slides: Implications for Bacterial Capture on Antibody Microarrays

Campylobacter is a major cause of foodborne illnesses worldwide. Campylobacter infections, commonly caused by ingestion of undercooked poultry and meat products, can lead to gastroenteritis and chronic reactive arthritis in humans. Whole genome sequencing (WGS) is a powerful technology that provides comprehensive genetic information about bacteria and is increasingly being applied to study foodborne pathogens: e.g., evolution, epidemiology/outbreak investigation, and detection. Herein we report the complete genome sequence of Campylobacter coli strain YH502 isolated from retail chicken in the United States. WGS, de novo assembly, and annotation of the genome revealed a chromosome of 1,718,974 bp and a mega-plasmid (pCOS502) of 125,964 bp. GC content of the genome was 31.2% with 1931 coding sequences and 53 non-coding RNAs. Multiple virulence factors including a plasmid-borne type VI secretion system and antimicrobial resistance genes (beta-lactams, fluoroquinolones, and aminoglycoside) were found. The presence of T6SS in a mobile genetic element (plasmid) suggests plausible horizontal transfer of these virulence genes to other organisms. The C. coli YH502 genome also harbors CRISPR sequences and associated proteins. Phylogenetic analysis based on average nucleotide identity and single nucleotide polymorphisms identified closely related C. coli genomes available in the NCBI database. Taken together, the analyzed genomic data of this potentially virulent strain of C. coli will facilitate further understanding of this important foodborne pathogen most likely leading to better control strategies. The chromosome and plasmid sequences of C. coli YH502 have been deposited in GenBank under the accession numbers CP018900.1 and CP018901.1, respectively.

An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules

The thixotropic-like properties of saline/glycerol drops, containing biotinylated capture antibodies, on streptavidin-coated glass slides have been investigated, along with their implications for bacterial detection in a fluorescent microarray immunoassay. The thixotropic-like nature of 60:40 saline-glycerol semisolid droplets (with differing amounts of antibodies) was observed when bacteria were captured, and their presence detected using a fluorescently-labeled antibody. Semisolid, gel-like drops of biotinylated capture antibody became liquefied and moved, and then returned to semisolid state, during the normal immunoassay procedures for bacterial capture and detection. Streaking patterns were observed that indicated thixotropic-like characteristics, and this appeared to have allowed excess biotinylated capture antibody to participate in bacterial capture and detection. When developing a microarray for bacterial detection, this must be considered for optimization. For example, with the appropriate concentration of antibody (in this study, 0.125 ng/nL), spots with increased diameter at the point of contact printing (and almost no streaking) were produced, resulting in a maximal signal. With capture antibody concentrations greater than 0.125 ng/nL, the excess biotinylated capture antibody (i.e., that which was residing in the three-dimensional, semisolid droplet space above the surface) was utilized to capture more bacteria. Similarly, when the immunoassay was performed within a hydrophobic barrier (i.e., without a coverslip), brighter spots with increased signal were observed. In addition, when higher concentrations of cells (∼108 cells/mL) were available for capture, the importance of unbound capture antibody in the semisolid droplets became apparent because washing off the excess, unbound biotinylated capture antibody before the immunoassay was performed reduced the signal intensity by nearly 50%. This reduction in signal was not observed with lower concentrations of cells (∼106 cells/mL). With increased volumes of capture antibody, abnormal spots were visualized, along with decreased signal intensity, after bacterial detection, indicating that the increased droplet volume detrimentally affected the immunoassay.