Development of green fluorescent protein-expressing bacterial strains and evaluation for potential use as positive controls in sample analyses.

Strains of enterohemorrhagic Escherichia coli O157:H7 and Salmonella Typhimurium were engineered to express the gene for a modified green fluorescent protein (GFP) and were evaluated for potential use as positive controls in sample analyses. The strains fluoresced when observed as colonies with a handheld UV lamp or as individual cells under a fluorescent microscope. The strains maintained their fluorescence following growth in three series of transfer experiments including 8 to 11 passages from broth to broth and twice for 15 consecutive transfers from broth onto Trypticase soy agar plates. Cultures also maintained stability in the ability to fluoresce when agar plates were refrigerated (4 degrees C) for up to 12 days. Growth characteristics of the GFP-positive strains were comparable to those of corresponding control strains. The GFP-positive strains were successfully identified using rapid diagnostic methods and were differentiated from their corresponding non-GFP strains by pulsed-field gel electrophoresis but not by repetitive extragenic palindromic PCR. The GFP-positive and the control strains were recovered successfully from individually inoculated food samples (Feta cheese, raw shrimp, cooked shrimp, and cooked crawfish). However, in one Feta cheese sample and one raw shrimp sample inoculated with combined GFP-positive and GFP-negative cultures, colonies of the GFP-positive strains were not observed under UV light; fluorescing cells in one of the inoculated samples (raw shrimp) were revealed by microscopy. In general, the isolates from the inoculated foods were GFP positive by microscopic examination; the pure isolates could also be restreaked onto Trypticase soy agar, and colonies could be visually examined under UV light. Because GFP strains are not known to occur naturally in the environment, the use of the Salmonella GFP-positive strain may offer advantages as a positive control even when distinct and rare serotypes are available. The GFP-positive E. coli O157:H7 strain may also prove beneficial for use as a positive control strain for sample analyses.

Production and Characterization of Monoclonal Antibodies To Detect Vibrio cholerae Serogroup O1 in a Rapid Enzyme-Linked Immunosorbent Assay.

Monoclonal antibodies (MAbs) were developed for a rapid and efficient screening procedure to detect cultures of Vibrio cholerae serogroup O1. Spleen cells of BALB/c mice previously immunized with an attenuated control strain of V. cholerae were fused with mouse myeloma cell line SP2/0. An enzyme-linked immunosorbent assay (ELISA) was used to test cultural hybridoma secretions of two MAbs against 120 strains of V. cholerae O1, 38 strains of V. cholerae non-O1, 15 strains of other Vibrio spp., and 20 strains of other bacterial species. Results of tests using both MAbs were identical. The MAbs successfully detected all of the confirmed serotype O1 strains. Three additional V. cholerae strains that agglutinated antisera and the saline control were considered serologically inconclusive. Of these, one was detected as positive for V. cholerae by both MAbs. The MAbs gave no false-positive reactions when tested against the confirmed non-O1 strains, other Vibrio spp., and other bacterial species. Use of this ELISA will enhance the speed and accuracy needed for detecting V. cholerae O1 cultures.

Detection of Listeria spp. in Naturally Contaminated Seafoods Using Four Enrichment Procedures.

Four enrichment procedures were evaluated for the recovery of Listeria spp. from 211 samples of raw and processed seafoods. The presence of Listeria spp. was determined in all four methods by a commercial enzyme-linked immunosorbent assay kit. The enrichments used were 1) Listeria enrichment broth (LEB); 2) buffered LEB (BLEB); 3) BLEB transferred to the same enrichment after 24 h (BLEB 24-h transfer); and 4) modified University of Vermont medium (UVM-1) transferred after 24 h to UVM-1 medium containing additional acriflavin (UVM-2). All four enrichments were incubated for a total of 48 h at 30°C. To determine the efficiency of each protocol, we compared our recovery results with those obtained by using a modified version of the Bacteriological Analytical Manual (BAM) cultural method, as described in the Federal Register of November 1, 1988. Statistical analysis showed that recovery of Listeria spp. using nonbuffered LEB for 48 h without transfer did not differ significantly from that obtained with the revised BAM method.