Identification of Toadfish-Pathogenic Bacteria Based on a Comparative Molecular Approach.

Abstract

Opsanus tau (oyster toadfish) is an important laboratory animal at the Marine Biological Laboratory (MBL); they are used as a model for studying the mechanisms of hearing and balance. A new disease, bacterial pericarditis, which is caused by a gram negative bacterium, has caused mortalities in the Marine Resources Center (MRC). Determining the identity of this pathogen, which has not been previously seen in toadfish (1), is important to the health management program for this species. A rapid and accurate method, based on the 16s rRNA gene, for the identification of bacterial pathogens was used to address this problem (2, 3). Bacterial strains (n = 15) were isolated from individual infected toadfishes (n = 12) during the outbreak of bacterial pericarditis that occurred from July 1996 to April 1997 at the MRC. Brain heart infusion agar (BHIA; Difco, Detroit, MI) with 1.2% NaCl, was used to isolate and purify bacterial cultures. All isolates were first tested for antibiotic resistance. BHIA antibiotic plates, including ampicillin (50 pg/ml), kanamycin (25 pg/ ml), tetracycline (10 pg/ml), and vancomycin (100 pg/ml), were used. The plates were incubated for 24 h at room temperature. Bacterial DNA was isolated from each isolate using the bead beater procedure and standard phenol extraction. Universal prokaryote primers specific to the ends of prokaryotic 16s rRNA were used to amplify this gene. DNA hybridization (checker board) was applied to the 16s rRNA of all isolates using 16s rRNA complementary probes: universal prokaryotic, enteric, betas, deltas, sulfate reducing bacteria, flavobacteria, gram positive low GC, and spirochete. To screen the PCR products and cornFare the 16s rDNA in all isolates, restriction fragment length polymorphism analysis (RFLP) was performed using four tetrameric restriction enzymes, HaeIII, HinPI, MspI, and RsaI (4). To confirm the results obtained from the RFLP analysis, denaturing gradient gel electrophoresis (DGGE) was applied. DGGE primers were used to amplify the 16s rRNA gene, and the PCR products were run in a 40%-60% urea formamide gradient acrylamide gel. After the RFLP and DGGE comparisons, representative samples with a common RFLP DNA pattern and DGGE band positions were chosen for sequencing with an ABl automatic apparatus. Sequences obtained were compared to the non-redundant nucleotide database at the National Center for Biotechnology Information using the BLAST (Basic Local Alignment Tool) algorithm. Phylogenetic analyses were done according to Paster (5). All isolates (#I -#15) tested for antibiotic resistance were susceptible to ampicillin, kanamycin, and tetracycline, and were resistant to vancomycin. If antibiotic treatment is needed, ampi-