Thirty-four strains of nonfermentative, respiratory, luminous bacteria were isolated from samples of squid ink and seawater from depths of 200 to 300 m in the Alboran Sea. Although these strains had a few properties similar to properties of Shewanella (Alteromonas) hanedui, they did not cluster phenotypically with any previously described bacterium. The nucleotide sequence of a 740-bp segment of l d was not homologous with other known l d sequences but clustered with the 2 4 sequences of Shewanella hanedai, fibrio logei, fibriofischeri, and Photobacterium species. The 16s RNA gene from two strains was sequenced and was found to be most closely related to the S. hunedai 16s RNA gene. Based on the differences observed, we describe the new isolates as members of a new species, Shewanella woodyi sp. nov. Strain ATCC 51908 (= MS32) is the type strain of this new species.The first isolates of exclusively respiratory (nonfermentative) marine luminous bacteria were assigned to Alteromonas hanedai (2, 5a) and later were reassigned to the genus Shewanella (5b, 9). The genus Shewanella is composed of four species, and only one of these species, Shewanella hanedai, is bioluminescent. Compared to species of the genera Alteromonas, Pseudomonas, and Deleya, S. hanedai and Shewanella putrefaciens have unique quinone compositions, and these organisms possess menaquniones that are not present in Alteromonas species (1). Further confirmation that these Shewanella species form an independent clade that can be recognized as a genus came from 16s rRNA sequence analysis (4).Water samples and squid ink samples from intermediatedepth dives of the submersible Johnson Sea Link contained luminous bacteria that were found to be unable to ferment sugars but could grow anaerobically with nitrate or nitrite as a terminal electron acceptor. Because these organisms did not seem to be similar to S. hanedai, we performed a more complete phenotypic characterization along with a hybridization and sequence analysis of a 740-bp section of the luxA gene, and we also sequenced the 16s RNA gene from two strains. We designated these new isolates Shewanella woodyi sp. nov. The species name is in honor of J. Woodland Hastings, known to most as Woody, an American biologist who, among other things, has made numerous and valuable contributions to our understanding of the biochemistry and function of bacterial bioluminescence. MATERIALS AND METHODSAll of the strains analyzed in this study (Table 1) Our previous experience with luminous marine bacteria indicated that this medium was suitable for most luminous marine bacteria; however, S. woodyi grew but did not produce luminescence in GMA broth (this was later shown to be due to a salt requirement for luminescence). We also found that the strains whose designations begin with MS (MS strains) did not utilize glycerol. Consequently, the medium used for these strains was changed to Shewanella marine agar (SMA) containing (per liter) 5 g of Bacto Peptone (Difco), 1 g of Bacto Yeast Extract (Difco), 15 g of Bacto Ag...

An oligonucleotide DNA probe (VYAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hlyA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with WAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100%/Y specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by WAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 102 and 2 x 101 bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by WAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The WAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests.

A gas chromatographic method using a capillary column for rapid identification of Vibrio vulnificus was examined in a collaborative study. Identifications were performed by analysis of cellular fatty acid profiles which were automatically searched against reference profiles stored in a computer-generated library. Each of the 13 collaborators was sent 15 unknown isolates, which included 10 V. vulnificus isolates and 5 negative control isolates. Each collaborator was furnished with a computergenerated library, developed by the Dallas U.S. Food and Drug Administration laboratory, which contained entries for V. vulnificus, V. cholerae, V. fluvialis, V. parahaemolyticus, V. mimicus, and Aeromonas hydrophila. Of the 195 isolates sent to the collaborators, results for 190 isolates were received. The other 5 isolates were nonviable before analyses began. Of the 126 V. vulnificus isolates analyzed, 118 (93.7%) were correctly identified. Of the 65 negative control isolates sent, one was nonviable, one was misidentified as V. vulnificus, and 2 were misidentified as V. parahaemolyticus. Of the 64 negative controls analyzed, 95.3% were correctly identified. Statistical analysis shows a sensitivity rate of 0.872, specificity rate of 0.982, false positive rate of 0.010, and false negative rate of 0.206. The gas chromatographic method for identification of Vibrio vulnificus by microbial fatty acid profile has been adopted first action by AOAC INTERNATIONAL.

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