INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,OCt. 1986, p. 531-543 Vol. 36, No. 4 OO2@7713/86/O40531- 13$02. OO/O Copyright 0 1986, International Union of Microbiological Societies

Numerical of Isolated from Aquatic Environments P. A. WEST,lT P. R. BRAYTON,' T. N. BRYANT,*$ AND R. R. COLWELL1* Department of Microbiology, University of Maryland, College Park, Maryland 20742, and Department of Microbiology, University of Surrey, Guildford GU25XH, England2

A total of 443 strains of , many of which were isolated from estuaries in the United States, were studied with numerical taxonpmy to determe relationships among those species known to be pathogenic for humans, e.g., V. cholerae, V.mimicus, V.jluviulis, V.parahaernolyticus, and V. vulnijicus, as well as among strains not identifiable from the minimum diagnostic characteristics likely to be used in many clinical laboratories. Similarity coefficients were calculated on the basis of 133 characters, using Euclidean distance. Most of the strains clustered in phena along with reference strains. Several new, as yet undescribed phena containing strains isolated from diverse geographic locations were detected. Deficiencies in curreqt schemes for identification of V. vulnijicus and V.parahaemolyticus were observed, and the definitions of these species were extended by using information obtained in this study.

The genus Vibrio is currently classified in the family MATERIALS AND METHODS together with related genera Aeromonas, Plesiomonas, and Photobacterium (3, 4). Strains of the Bacterial strains. The 443 strains included in this study are genus Vibrio are straight or curved, gram-negative, rod- listed in Table 1. All strains were presumptively identified as shaped ; they are motile in liquid medium by means Vibrio or Aeromovas species at laboratories in each of the of a polar flagellum, capable of fermenting glucose study regions. A common identification scheme was adopted anaerogenically, and usually oxidase positive (35,46). Mem- by each laboratory, and methods for screening of the isolates bers of the genus Vibrio are ubiquitous in estuarine and are described elsewhere (34). The phenotypic characteristics marine waters and sediment and are associated with nitrogen used for presumptive identification of these organisms in- fixation (43, degradation of specific organic pollutants (48), cluded oxidase reaction; decarboxylation of L-arginine, L- and colonization of surfaces and internal organs of inverte- lysine and L-ornithine; glucose fermentation; H2S produc- brate and vertebrate marine animals (10, 22). Under condi- tion; indole productian; o-nitrophenyl-P-D-galac- tions of stress, some of the species that are commensal to topyranoside (ONPG) hydrolysis; growth in 0 and 7% NaCl; marine animals can act as pathogens, causing severe eco- sensitivity to Oh29 (10 and 150 Fg); gelatinase activity; and nomic loss for the mariculture trade (38). anaerogenic fermentation of lactose, sucrose, mannitol, D- Some Vibrio species are also pathogenic for humans (6), mannose, and L-arabinose. Most of the isolates were ob- and recent studies have revealed that these pathogens are tained from enrichments in alkaline peptone water, followed endemic in some coastal areas of the United States. Further- by inoculation onto thiosulfate-citrate-bile salts-sucrose agar more, the endemicity of Vibrio-related diseases is more (Oxoid, Columbia, Md.). Selected reference strains that likely due to the persistence of the pathogens in their natural represented species, biovars, and type strains of the aquatic reservoir rather than to the maintenance of the Vibionaceae were included in the study and are listed in disease in the human community (9, 25). Table 1. All strains were maintained on a solution contain- In this paper we describe the phenotypic characteristics of ing, per liter, 10 g of tryptone, 10 g of NaCl, 4 g of strains of the genus Vibrio that were isolated from areas in MgC12 - 6H20, 1 g of KCl, and 15 g of agar. the United States which are of importance for commercial Characterization tests. Methods for carrying out the phe- harvesting of fish and shellfish, as well as for recreational notypic characterization tests listed in Table 2 were de- use. The areas included the Chesapeake Bay in Maryland, scribed in detail previously (46, 47). These were used with- Appalachicola Bay in Florida, bayous in the Gulf of Mexico out m9dification. The probability of an erroneous two-state along the coast of Louisiana, and the shorelines and bays of character result was estimated by the procedure of Sneath the Pacific Northwest in Oregon. Also included for compar- and Johnson (39). ison were strains isolated frop Bangladesh and Puerto Rico. Analysis of results. Quantitative and qualitative data were This work was undertaken to investigate the ecology and coded and analyzed with a Prime 750 computer system and taxonomy of Strains of the Vibrionaceae from geographically the IBM podel 3741 computer. A similarity matrix was diverse origins. One objective of this work was to determine calculated with Euclidean distance, and a phenogram was the efficiency of the commonly used minimum set of char- constructed with the Jaccard coefficient and unweighted- acteristics for differentiation of Vibrio species (21, 35, 44). pair-group arithmetic average sorting. Procedures for deter- mination of the cophenetic correlation and best diagnostic characteristic for each phenon are described elsewhere in detail (45). * Corresponding author. t Present address: Aquatic Environment Protection Division 2, Ministry of Agriculture, Fisheries and Food, Burnham-on-Crouch, RESULTS AND DISCUSSION Essex CMOSHA, England. $ Present address: Faculty of Medicine, Southampton General Data matrix. Each strain was examined for 139 characters. Hospital, Southampton SO94XY, England. All strains were gram negative, oxidase positive, and able to

53 1 532 WEST ET AL. INT. J. SYST. BACTERIOL.

TABLE 1. Designation and source of strains assigned to phena derived from analysis by Euclidean distance unweighted-pair-group arithmetic average sorting Phenon no. and name Strain no. Source or site of Isolate 1. V.fluvialis W468 Water, Bangladesh W117 Human , Indonesia W69, W71, W72, W76, W321 Water, Chesapeake Bay, Md. W61 Yaquina Bay, Oreg. W211 Water, Oreg. W90 Sediment, La. W77, W78, W79, W80, W81, W82, W83, Water, La. W85, W86, dup. W86, W87 wga, ~89,w91 Water, sediment, plankton, Mud Lake, La. w93 Water, Sabine Lake, La. ATCC 33809T Human feces

2. V.furnissii W67, W68, W73, W74, W324 Water, Chesapeake Bay, Md. W114 Human gastroenteritis, Indonesia W92 Water, La. NCTC 11328 River water, England

3. V. natriegens W70, dup. W70, W75, W344 Water, Chesapeake Bay, Md. W361 Sediment, Chesapeake Bay, Md. W362 Oysters, Chesapeake Bay, Md. W387 Water, Yaquina Bay, Oreg. W285 Sediment, Yaquina Bay, Oreg. W396 Oysters, England W66 Water, Ha. ATCC 1404aT Marsh

4. V.parahaemolyticus w470, wm,w475, w476 wm,~480, Water, Bangladesh W481 W226, W265, W266, W267, W268, W269, Water, Chesapeake Bay, Md. W270, W272, W317, W319 W350 Oyster, Chesapeake Bay, Md. W63, W230, dup. W230, W273, W274 Water, Appalachicola Bay, Fla. W238, W245, W252, W253 Water, Mud Lake, La. W201, W248, W254 Sediment, Mud Lake, La. W235, W246 Crab meat, Mud Lake, La. W241, W242, W243, W244, W249, W251, Water, La. ~255,~256, ~257, ~258, w259 W275, W276, W281, W282, W287, W288 Sediment, Tillamook Bay, Oreg. W277, W280 Clams, Tillamook Bay, Oreg. W279 Crabs, Tillamook Bay, Oreg. W284 Water, Tillamook Bay, Oreg. w2a3 Water, Yaquina Bay, Oreg. W286 Clams, Yaquina Bay, Oreg. W 195 Seafood, Japan W191, W192 Human gastroenteritis w193 Unknown ATCC 17802T Food poisoning

5. V. alginolyticus W335, W472 Water, Bangladesh W64, W65 ' Water, Appalachicola Bay, Fla. W94 Feces ATCC 17749= Spoiled mackerel

6. Unidentified marine Vibrio spp. w353 Water, Tillamook River, Oreg. W432 Sediment, Garibaldi River, Oreg. w3a4, w388 Water, Oreg. W383, W385 Clams, Oreg. W390, W392 Brackish water ditch, England W389 Fresh water stream, England W433 Water, Chesapeake Bay, Md.

7. V. cholerae W31, W32, W33, W34, W35, W37, W38, Water, Chesapeake Bay, Md. w39, W44, Wl0l W152 Crayfish, La. ~150,wia, win, win,wm, ~177 Water and crabs, La. W8, W10, W42, W43, W127, dup. W127, Water, La. W144, W 145, W 146, W153, W199, W214, W215 Continued an following page VOL. 36, 1986 AQUATIC VIBRIOS 533

TABLE 1-Continued Phenon no. and name Strain no. Source or site of Isolate W128, W129, W130, W132, W133, W134, Water, plankton, sediment, La. W135, W136, W137, W138, W139, W140, W141, W142, W203 w3, w97 Water, Mud Lake, La. W2, dup. W2, W5, W9 Sediment, Mud Lake, La. W%, w109 Sewer, La. w4 Sewage, Lake Charles, La. w7 Water, Burns Pt., La., w11 Water, Lake Pontchartrain, La. W13 Sewage, New Orleans, La. W24 Water, Fla. W 148 Clinical isolate, Fla. W17, W18, W19, W20, W21, W23, W25, Appalachicola Bay, Fla. W26, W27, W28, W30 W41 Oyster, Ha. W46, W47, W50, W55, W56, W58 Water, Tillamook Bay, Oreg. w57, w59, w60 Sediment, Tillamook Bay, Oreg. W48, w49 Water, Coos Bay, Oreg. W51, W52, W54 Water, Yaquina Bay, Oreg. w53 Water, Oreg. W113, W437, W438, W439, W440 Ditch water, England Wlll Sediment, England W159, W167, W170, W173, W176 Clinical isolate W122, ATCC 14035T Human feces W99, W100, W108, W109, W110, W126 Human gastroentertitis W161, W169 Environmental isolate W104, W106, W149, W156, W157, W160, Unknown W171

8. V. mimicus W151 Crayfish, La. W102, W105, W154, W155 Feces, La. W6, W14, W147 Water, La. w 12 Water, Lake Pontchartrain, La. w1 Water, Calcasieu River, La. W29 Appalachicola Bay, Fla. W40 Water, Ha. w166 Human gastroenteritis W162 Unknown ATCC 33653= Clinical isolate

9. V. vulnijcus W216, W219, W220, W221, W224, W225, Water, Chesapeake Bay, Md. W320, W322 W36 Sediment, Chesapeake Bay, Md. w45 Crab, Chesapeake Bay, Md. W227 Clams, Tillamook Bay, Oreg. W231, W232, W234 Appalachicola Bay, Fla. W228 Water, Yaquina Bay, Oreg. W229 Crab intestine, Oreg. W239, W236 Water, Mud Lake, La. W247 Crab meat, Mud Lake, La. W237, W250 Sediment, Mud Lake, La. W260, W261 Water, La. W263 Sediment, Lake Pontchartrain, La. w264 Sewage, New Orleans, La. W178, W181, W183, W184, W212, W240 Unknown W180, W182 Clinical isolate

10. V. anguillarum w374 Water, Chesapeake Bay, Md. W84 Water, La. W441, W442, W443, W444, W445, W446, Oysters, La. W447, W448, W450 W206, W207, W209 Water, Oreg. W62 Oyster, Tillamook Bay, Oreg. ATCC 19264= Diseased fish

11. Unidentified marine Vibrio spp. W202, W204, W208, W210, W213 Water, plankton, sediment, Oreg. W449, W451, W452, W453, W454, W455 Oyster, La. Continued on following page 534 WEST ET AL. INT. J. SYST. BACTERIOL.

TABLE &Continued Phenon no. and name Strain no. Source or site of Isolate 12. A. hydrophila w22 Appalachicola Bay, Fla. W143 Water, La. ATCC 15467 Oil emulsion W378 Sewer, La.

13. V. tubiashii W343, W359, W368, W377 Water, Chesapeake Bay, Md. W367 Oysters, Chesapeake Bay, Md. W428 Water, Florida-Puerto Rico transect

14. Unidentified marine Vibro spp. W376, W360, W370, W371, W373 Water, Chesapeake Bay, Md.

15. Unidentified marine Vibrio spp. w205 Water, Oreg. w349 Water, Mud Lake, Oreg. W386 Water, Coos Bay, Oreg. W398 Water, Chesapeake Bay, Md. W417 Water, Puerto Rico W391 Brackish water ditch, England

16. V. pelagius W411, W487, W490 Water, Puerto Rico ATCC 25916 Seawater

17. V. diazotrophicus ATCC 33466T Sea urchin W380 Water, La.

18. Aeromonas spp. w333 Water, Chesapeake Bay, Md. W233 Appalachicola Bay, Fla. W435, W436 Freshwater stream, England ATCC 7966T, dup. ATCC 7966T Milk

19. V. splendidus W218, w222 Water, Chesapeake Bay, Md. W413, W424 Water, Florida-Puerto Rico transect ATCC 33125T Marine fish W158, W164, W382 Unknown 20. V. campbellii W291, W293, W295, W296, W301, W303, Water, Florida-herto Rico transect W305, W306, W309, W312, W314, W315, W399, W403, W405, W407, W408, W409, W410, W412, W414, W415, W416, W418, W420, W422, W423, W434 W482, W484, W485, W486, W488, W489, Water, Puerto Rico W491 ATCC 25920T Seawater

21. Unidentified marine Vibrio spp. W290, W294, W298, W302, W304, W307, Water, Florida-Puerto transect W310

22. Unidentified marine Aeromonas-like w345, w346, W348 Water, Chesapeake Bay, Md. SPP . 23. Plesiomonas shigelloides ATCC 14029T Clinical isolate ATCC 14030 Clinical isolate

Unclustered strains W271, W316, W318, W323, W325, W326, Water, Chesapeake Bay, Md. W327, W330, W357, W358, W364, W366 W356 Sediment, Chesapeake Bay, Md. w95 Bird, cow, animal feces, Mud Lake, La. W351 Sediment, Mud Lake, La. W352 Water, Mud Lake, La. w200 Water, La. W278 Sediment, Oreg. W233 Appalachicola Bay, Ha. W381 Sediment, La. W372, W469, W471, W473, W474, W479 Water, Bangladesh dup. W417 Water, Puerto Rico W421, W404 Water, Florida-Puerto Rico transect W465, W466 Oyster w397 Sea urchin V. harveyi ATCC 2591gT Amphipod Continued on following page VOL.36, 1986 AQUATIC VIBRIOS 535

TABLE I-Continued Phenon no. and name Strain no. Source or site of Isolate V. albensis ATCC 14547T Fish V.proteolyticus ATCC 1533gT Intestine, Limnoria tripunctata V. nigrapulchritudo ATCC 27043= Seawater V. vulniJicus ATCC 27562= Blood V. nereis ATCC 25917= Seawater V. splendidus ATCC 25914 Seawater V.jischeri ATCC 774tT Seawater W179, W379 Unknown V. damsela W431 Infected damselfish ATCC, American Type Culture Collection, Rockville, Md.; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, England; W, culture collections of P.A.W., University of Maryland, College Park; T, type strain; dup., duplicate.

ferment glucose. All strains grew at pH 10.0 and failed to ganic compounds and in the overall nitrogen cycle in marine grow on butyrate and valerate as sole carbon sources. These environments is yet to be resolved. Four strains which six characteristics were excluded from the final data matrix, clustered in phenon 3 were originally identified as presump- since they were not of differentiating value. Taxonomic tive V.fluvialis. This suggests that there was a temporary resemblance among the 443 strains examined was accord- deficiency in the arginine decarboxylase test used by one ingly based on 133 characters. The probability for test error laboratory when these strains were first examined. was estimated to be 5.4% by the method of Sneath and Phenon 4 contained 60 strains identified as V. para- Johnson (39). haemolyticus. The ecology and pathogenicity of this species Description of phena. Taxonomic relationships derived have been extensively studied since the 1950s, when this from Euclidean distance and UPGMA sorting are illustrated organism was first reported to be associated with seafood- in Fig. 1. Euclidean distance was transformed into percent- borne human disease. The frequent occurrence of this orga- age of similarity (%S). The cophenetic correlation coefficient nism in estuarine waters of the United States is well estab- associated with the phenogram was 0.837. Twenty-three lished (23,24). However, it is disconcerting to note that of 48 phena included all but 43 strains. Table 2 lists characteristics fresh isolates received from each study area in the United of each phenon as well as those characteristics distinguishing States 11 (23%) were misidentified, since they did not fall the phena. into phenon 4. Of these 11 strains, 7 clustered with phenon 9 Phenon 1 formed at 89.7%S and contained strains of (V. vuln$cus). In addition, nine isolates in the phenon were Vibriofluvialis.All of the isolates except the reference strain received under a variety of other presumptive identifications in the phenon were correctly identified by the limited num- (five were identified as V. vuln$cus, three were identified by ber of tests. Two biovars were reported in the original an unknown designation, and one was identified as V. description of this species (26). Subsequent taxonomic in- fluvialis).This situation clearly indicates a serious deficiency vestigations of the biovars of V. fluvialis demonstrated in the present abbreviated schemes for differentiation of the sufficient genotypic dissimilarity to warrant separate two closely related species, V. parahaernolyticus and V. speciation (8). Accordingly, V.fluvialis made up biovar I, vuln$cus. Some of the characteristics considered essential and biovar I1 was created for V. furnissii. V. fluvialis for identification of V. parahaemolyticus-such as growth at characteristically ferments glucose anaerogenically and is 42"C, no growth in the presence of 10% NaC1, and inability frequently isolated from clinical diarrheal infections, espe- to ferment sucrose-are shown in the present study to have cially following consumption of raw or undercooked seafood an unacceptably high degree of variability when used with (41). The epidemiology of this disease is poorly understood strains freshly isolated from the environment. and detailed reports of the ecology of V.fluvialis are limited Of the six strains clustered in phenon 5, which is consti- to Louisiana coastal water (1). We report here the isolation tuted of the species V. alginolyticus, four were received as of this species from coastal and estuarine waters of Oregon, unidentified, casting doubt on the value of the short identi- the Chesapeake Bay, and Bangladesh. Strains of V.fluvialis fication scheme used by each laboratory. Other key diagnos- superficially resemble other arginine-decarboxylase-positive tic characteristics, such as swarming, positive Voges- members of the Vibrionaceae, such as V. anguillarum, and Proskauer reaction, and growth in presence of 10% NaCl, Aeromonas hydrophila. Nevertheless, the common scheme should be included in schemes for identifying this organism. used by each laboratory in this study was effective for Strains in phenon 6 were received as isolates of unknown distinguishing among these three species with a limited identity and could not be assigned to a known species or number of taxonomic tests. biovar within the Vibrionaceae even after extensive pheno- Phenon 2 contained eight strains of V.furnissii. All wild typic characterization. All strains failed to decarboxylate isolates received for the present study were presumptively L-arginine, L-lysine, and L-ornithine, and 2 isolates were identified as members of biovar I1 of V. fluvialis. The shown to fix nitrogen (45). We conclude that they represent ecology and potential pathogenicity of V.furnissii is poorly a new species. understood, despite its infrequent association with human Phenon 7 clustered at 85.5%S and contained 115 strains of disease (19). However, we are able to confirm the presence V. cholerae. There has been increasing interest in this of V.furnissii in Louisiana and Maryland estuarine waters. species in the United States, after outbreaks of in Phenon 3, clustering at 86.3%S, was identified as V. areas where the disease had been absent for many decades natriegens. Recent studies show that some strains of V. (5). Several studies report the widespread occurrence of this natriegens included in this study can fix nitrogen (45). The organism in estuarine waters of the United States (9). role of V. natriegens in mineralization of organic and inor- Serologic studies of V. cholerae establish that one serovar, 536 WEST ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Characteristics of phena identified in this study No. of positive strains'

h 2

h 5 h h h \o 5 v 2 d d b d 4 4 s! 3 E Phenon no. an0 name 2 (no. of strains)

h

h n z 3 h m 3 h d n 2 s 4 3 s s s s s s s 4 s ci 0; rl ci mCI m i Ir; od 0; d 5 d N N Growth in: 0% NaCl 31 -b-- - 114 14 - - - + ----- +--- ++ 6% NaCl + + + + + + 23 227 + + 12 4 5 3 + 2 6 + + + 1 8% NaCl 257 158+ 7- -- I----- 11-- I--- 10% NaCl _-- 55 _------Growth at: 4°C ------1-- 15 + 2 - - - - - +29--- 10°C 25 4 114 + + 106 13 6 + + + + 2 3 - + + 4 - - + + 35°C + + + + + + + + + + + + + + + 3 + + 4 32 1 + + 42°C 10 - - 38 1 - 111 13 24 - - 2 - - 3 1 - - 2 2 - + + Nitrate reduction +++++++++++++++++++311++ IJrease" -- 8 15 - 3 - - 2 - - - 3-- 1--2-- +- ONPGd ++ 86-++14+++++-+4++6---- H2S production _------1 -___-_------1 Voges-Proskauer -- - 1+-103 1-+1------+2---- Motility ...... Indole 255 -++2+++15+++4+2+++34- +1 Ciluconate oxidation -- - 1--32-- 11 2 ------4----- Arginine decarbox- ++ ------+++++--1+3-- -+ YlW Lysine decarbox- -- - 59 5 - + + + - 8 2 - - - - - +47--+ ylase Clrnithine decarbox- -- - +4-+ +321 _-_-_--- 4-- -+ ylase Thornley's arginine ++ 11-- 1-- +++++--+5 1---+ Growth at pH 4.5 11 - 2 ------I--- ++ Chick cell aggluti- -- 4 2 - 2 97 5 10 15 + + - - 2 - - 417--1 nation Hemolysis 83- 7--921413222--21-21-1-- L,uminescence ------11 - 2 ------Catalase + +. + + + + + + + + + + + + + + + + + 35 + + + Swarming -- -4958 - -_-_-_-_---_-___ Gas from glucose -+ ------2-----5---- 1 Acid from: Arbutin 22- + --- 10 8 32 2 1 - 3 + 2 - + + 2 - - + - Trehalose + + + 58+ + 112 + + + + + + + + + + + 6 + 4 - + Sucrose +++ 3+++ 2515+3++53++21--- Cellobiose 23 - 9 + + - 102 + 32 + 9 + + + 5 + + 1 + + - - - Mannose 22+ 4 + + - 80 + + + + + + + 5 + - + + 14 1 + + Inositol --7 ------1-1-+1 Mannitol + + + + + 9 106 + 21 + + + + 2 5 3 + + 3 13 1 + 1 Sorbitol 4- 3 ------12- - - - 3--2---- 1 Salicin 212+ 11- 71+-1-4+3-1+431-+- Ara binose + + + 50 - 9 - 12+ - 2 - - 2 - 4 5 - - - - - Resistance to: 01129 (10 pg) 25 + + 59 + 7 20 2 7 13 - + - - - 3 + + 3 26 3 + + 01129 (50 Fg) 116103631 1-18-+----- +-101+1 0/129 (150 pg) 13 -103------+----- +- 1- +- Novobiocin (5 p,g) 25 + 10 49 4 3 1 4 8 7 + + 3 133 + 5 2 - - ++ Continued on following page VOL. 36, 1986 AQUATIC VIBRIOS 537

TABLE 2-Continued No. of positive strainsa

n 2

n rl3 h n v 2 sW = d ti ti d 4 4 4 4 Phenon no. and name .QE (no. of strains) $

Q h \o W h n h n 8 2 m '$ ad 3 c2 3 W B B 3 5 .-rE .-3 a .Y Y U seo .f Q .-a" s E 3 x e s s s s s s s s 5 4 s 3Fl rl m v- r-: od 0; 5 5 5 Streptomycin(10p.g) 1 1 - 7 - - 2 1 20 5 4 1 - 3 - 1 - 1 3 7 - - + Ampicillin (10 pg) 24+ -++-12 14+ 52--1 2-4 4+4-- Polymyxin B (50 IU) 1--37--1021+---- 3 5 2 - 2 - 30 2 + - Brilliant green 4---15 ------1 (O.W2%) Crystal violet -- ---+-----1-----3 ----- (0.002%) Tellurite (0.0005%) 10 1 4 21- + 104 + 29 9 4 12- 4 - + - 2 - - - - Methylene Blue (0.01%) + + 6 3 - 1 - - - - - 1 - - - - - 3-12++ Methylviolet (0.0002%) + + 9 57 5 3 52 - 25 14 8 3 5 2 2 + - 5 5 35 3 + + Pyronin Y (0.002%) 13 - 627----- +----- +--- +1 Neutral red (0.002%) ++++++++32+++++++++++++1 Teepol 610 (0.4%) ++++++114++15+3++++++7++++ Bile salts (0.5%) ++ + ++ + + ++ ++ ++ ++ ++ + 735+ ++ Utilization as sole carbon source: Acetate + + + + + + 104 + 15 8 - 3 2 - - 2-1-1-++ Citrate ++++++106+31++3++5++5122-++ Fumarate ++ ++++112++++3++++++-354++ Glutarate I+ 3 1 ------DL-Glycerate + 6 10 58 + - 78 + 27 + + + + 4 - 1 - + - 10 - - + DL-3-Hydroxy-butyrate 25 + + 2 - + - - 1 - - - 2---- I----- a-Ketoglutarate + + + + + + 111 + 28 4 1 + - - 4 3 1 1 - 35 - - + DL-Lactate ++ 9++2 7122--2--2-- 1-2--+ Malate ++ 22049 1---- 1--2-- 2- 1-++ Malonate --+ ---- 1 __-__------Propionate ++10234+ 1------2------+ Succinate ++ 9+++12113-6+4-4+-51302++ D- Alanine + + + + + + 15 13 27 1 - 2 5 + 5 + 1 1 - +5+- L-Alanine + + + 59 + + 32 630 3 124 4 + 3 1 1 - 33 + + - Gammaamino butyrate + + + 1 ------24--2-2 ----- Delta amino valerate - 6 + - - 2 ------2 ------L- Arginine + + + + 5 + 19 9 28 12 6 3 4 + + + + 2 118 4 - - L- As paragine 25 + + + 4 8 105 + 27 6 7 3 2 3 4 + 14- 19 4 - + L- Aspartate + + 9 59 + + 12 8 + 11 10 3 + 3 4 3 - 3 - 12 2 - + L-Citrulline +2+545- 2591-121+++-- 12+-- L-Glutamate + 7 + + + + 89 14 26 + + 3 4 3 5 + + 4 - +5++ L-Glycine 195+57++ 148---- 4-+--- 75-- L-Histidine ++ ++++105+24++++3++++3174++ L-Hy drox yproline I- 150+-- -2--- 22------+- L-Leucine -- 753+2 _------I--- L-Ornithine + + + 55 5 + 4 4 16 3 5 15+ + - + - - 65-- L-Proline + + + 59 + 7 113 + 27 13 + 3 + + + + + 3 - 32 + + + L-Serine 25 + + 56 + + 34 6 9 6 3 + - - + 2 - 3 - 16 5 + + L-Threonine + + + 59+ + 9 932 3 7 3 + + + + 12- 34+ - - L-Tyrosine 246 671+- 66----- 4------+- D- Amygdalin +2 45-- 1 __--_------L- Arabinose ++ +521+- -- 11 - 1 - 3 1 - + 2 - 1 - + - Arbutin +5+ 4-1---1------4--- +- Continued 538 WEST ET AL. INT. J. SYST.BACTERIOL.

Phenon no. and name (no. of strains)

Cellobiose 9- +572- 11 4 + 15 + + + + 5 3 + 3 - 34 - - - r)-Fructose +++a+++ +31+++++++++- 94++ n-Galactose ++ +573+ 109 +31+++5-+++5- 4-++ 1)-Gluconate ++ 10 52+ + + +++6++4++++- 1- ++ D-Glucose ++++++ + +++++++++++-++++ Glycogen ++++++ 69 + + + + + + + 5 + + + - 35 - - + Lactose -- 11-- 1 -12--- 1-11-1 ----- Maltose ++++++109 + + + + + + + + + + + - 33 - - + D-Mannose 22+ 5 +4 - 77 14 + + + 3 + + 3 + - + - 11 - + + hlelibiose 2- 8 1-- -3---+-- I------+ Nibose ++lo+++ 18 + 30 14 + + + + + + + 5 - 33 1 + + Salicin ++ + 11-4 6 1322-344-- 5- 2- ++ Sucrose ++ + 16++ 112 6 6 15 + + + + 5 + + + - 17 - + + Threhalose ++++++114 + + + + + + + + + + + - 22 + 1 + Xylose -- - 1- 1 ------11--- -+ N-acetyl D-glucosamine ++++++ + + + + t + + + 5 + + + - 32 5 + + Ethanolamine -- - 1-+ - D-Glucosamine ++++5+ 110 + 31 + + 3 2 + 5 2 + 4 - - - ++ Putrescine 14+ + +-+ - -I--- 1-- +-I----- .4denine -- - I-+ - Glucuronate 221 334-- 29 Galacturonate 26+ 2--- - Sarcosinc 2- 6--- Xanthine -- I--- Taurine -- 4--- D-Arabitol 22+ + --- Dulcitol -- - 21- Ethanol ++ 75423 Glycerol ++++++ m-Inositol -- 7 1-- Mannitol ++++++ 1-Propanol +7 75529 Sorbitol 4- I--- p-Hydroxybenzoic wid ------Phenylacetate 1- - 1- 2 Production of: Amylase +7++5+101 -++++++++++++--- Gelatinase ++++++ + +++++++++-+6++-- Elastase -- --_6 85 225+ 1--- 5-- 4-21-- Lecithmase ++++++114 + + 15 + 1 + + 5 + - 4 6 + + - - Alginase _----- 1 --- 5---- 31------Deoxyribonuclease ++ 6++9 + +++++++++-++++-1 Phosphatase +7+59++ 96 1430+++++++++7+++- Sulfatase -- 1 l-- - 4 ------2 ------Chitinase ++-++9 + + 32 15 + 3 4 - 3 + - + + + 3 - - Chondroitinase -1 - 113 Degradation of: Albumin 146 - 95+114 8 28 + 8 2 1 1 + - - 4 3141-- Casein ++ 9+++ + + + + + + + + + + - + 5 33 3 - - Continued on following page VOL.36, 1986 AQUATIC VIBRIOS 539

TABLE 2-Continued No. of positive strainsa

h vm

Phenon no. and name (no. of strains)

tyrate and valerate. ' +, All strains positive; -, all strains negative. Tests in boldface type are useful for distinguishing phena. ONPG,o-nitrophenyl-P-D-galactopyranoside. designated 0-1, is most frequently associated with pandemic taxonomic studies have demonstrated the widespread occur- cholera, whereas other serovars, collectively designated rence of luminous non-0-group 1 serovars of V. cholerae. non-0-group 1, are commonly free-living in aquatic environ- Hybridization studies suggest that V. albensis is taxonami- ments (7, 12). Most wild isolates received were from the cally synonymous with V. cholerae (16). However, the level non-0-group 1serovar, and presumptive identifications of all of clustering of V. albensis ATCC 14547 with strains of V. of them proved correct. Only three strains received that cholerae in this study and previous investigations (13, 47) were of unknown identity were included in this cluster. suggests that strain ATCC 14547 is inappropriate as the These grew in 6% NaCl and were resistant to 01129 (10 phenotypic representative for luminous non-0-group 1 V. pg/ml) but gave other reactions consistent with V. cholerae. cholerae. Of the 115 strains of the V. cholerae phenon, 11 (10%) were Phenon 9 contained strains of V. vulnificus isolated from luminous, confirming conclusions from previous taxonomic aquatic environments and cases of clinical disease. V. studies that bioluminescence is a characteristic of this spe- vulnificus is an opportunistic human pathogen commonly cies, albeit variable (13, 16, 47). None of the 0-1 serovars associated with infections of wounds exposed to seawater or was luminous. with consumption of raw shellfish (40). The frequency of Strains of phenon 8 clustered at 88.8%S and were identi- infection is related to the abundance of the pathogen in its fied as V. mimicus. Strains of V. mimicus were historically natural aquatic habitat (9). Despite phenotypic resemblance known as group V V. cholerae strains according to the to other vibrios that do not ferment sucrose, the trait which grouping scheme of Heiberg (18), which was based on first drew attention to V. vulnificus was its ability to ferment fermentation of sucrose, D-mannose, and L-arabinose. lactose (20). Subseqent taxonomic studies have indicated Vibrio mimicus, named because of its similarity to V. chol- that V. vulnificus is easily confused with V. para- erae, was found to be associated with a diarrheal illness that haemolyticus. It is likely that several unusual clinical mani- sometimes resembled cholera, as well as with extraintestinal festations of infections historically associated with V. infections (36). The main feature used to distinguish V. parahaemolyticus were caused by V. vulnifcus (6). mimicus from V. cholerae is sucrose fermentation (11). In the present study, 7 of 48 strains (15%) presumptively Although strains of V. mimicus fail to ferment sucrose, identified as V. parahaemolyticus were identified as V. additional traits, such as negative Voges-Proskauer reaction vulnificus after further characterization. The confusion may and failure to produce extracellular amylase, should also be be caused by the reliance on the QNPG reaction and lactose demonstrated to ensure adequate differentiation from V. fermentation to distinguish V. vulnijcus from V. cholerae (14). parahaemolyticus and related marine vibrios. It has been V. albensis ATCC 14547 clustered at the edge of the V. suggested that lactose fermentation follows emergence of mimicus phenon and linked with phenon 7 (V. cholerae) at S mutants from lactose nonfermenting wild types during labo- L 83.0%. In Bergey 's Manual of Determinative Bacteriology ratory culture and isolation procedures (2). In addition, (8th ed.), this single strain is described as the luminescent lactose fermentation is now recognized as a trait common biotype of V. cholerae (37). Subsequent ecological and among marine and estuarine vibrios (32, 43). Biochemical 540 WEST ET AL. INT. J. SYST.BACTERIOL.

PERCENT SIMILARITY No. of 70 80 90 -I00 Phenon Strains Identification I I 1 26 V. f luvialis 2 8 V. furnissii f- 3 11 V. natriegens 4 60 V. parahaemdyticus a-5 6 V. alginolyticus 6 10 Vibrio spp.

7 115 V. cholerae

8 15 V.mimicus

9 33 V. vulnificus

10 16 V. anguillarum 11 11 Vibrio spp. 12 4 A.hydro hila 13 6 V: t "bias& 14 5 Vibrio spp. 15 6 Vibrio spp. ri 16 4 v.pelagius

17 2 V. diazot rophicus 18 6 Aeromonas spp. 19 8 V. splendidus P-

r 20 36 V. campbellii 1L I-- 11 4' 21 7 Vibrio spp. 22 3 Aeromonas-like spp. 23 2 I! shlgelbides

I 1 a I 60 70 80 90 100 FIG. 1. Phenogram showing relationships among phena based on the Euclidean distance coefficient and unweighted-pair-group arithmetic sorting with distances transformed into percentages of similarity. Unclustered strains, from top of dendrogram to bottom, are as follows: W323, W325, W471, W479, duplicate W417; W95, ATCC 25919, W233, W351, W278, W271, W356, W381, W352; ATCC 14547; W357; W364, ATCC 15338, W326, ATCC 27043, W200, W366, W379; W327, W330; W358, ATCC 7744, W431; W404, ATCC 27562, W466, W397, W318, W316; W465, ATCC 25917, ATCC 25914, W421, W179, W469; W372; W473, W474. VOL.36, 1986 AQUATIC VIBRIOS 541

tests commonly used by clinical laboratories to identify V. contained strains of Aeromonas that were most closely vulnificus are often time-consuming, whereas the rapid related to A. hydrophila biovar hydrophila (33). spread of symptoms and high mortality rate associated with Phena 19, 20, 21, and 22 included decarboxylase-negative severe infections of V. vulnijicus require rapid differential vibrios described in detail elsewhere (45). Phenon 23 was diagnosis. A serological rapid identification test (31) may identified as P lesiomonas shigelloides and contained only therefore be more appropriate than phenotypic characteriza- the type and reference strains. tion. Several type and reference strains remained unclustered, Sixteen strains identified as V. anguillarum clustered in notably V. vulnificus ATCC 27562T, which did not join the phenon 10. Two strains presumptively identified as V. cluster of strains constituting phenon 9. fluvialis also clustered in phenon 10, along with three argi- As noted above, all strains employed in this study were nine-positive strains and one decarboxylase-negative strain. presumptively identified with a set of tests selected to Strains of V. anguillarum are associated with vibriosis of differentiate the pathogenic vibrios, e.g., V. cholerae, V. fish, a septicemic disease causing sudden death in fish (38). fluvialis, V. vulnificus, and V.parahaemolyticus. This field However, there is considerable variation in phenotype operation was intended to eliminate time-consuming tests, among strains previously identified as V. anguillarum (47), such as testing growth on a large number of organic com- and this has led to creation of several new species within the pounds as the sole source of carbon. In addition to the genus for strains which superficially resemble V. anguil- presumptively identified strains, strains of unknown identity larum (3). The strain comprising phenon 10 was thus V. were also examined, and more extensive characterization anguillarum sensu stricto, since the type strain for the was carried out. It is clear that the scheme adopted for species, ATCC 19264, was included in the cluster. identification of V. cholerae, V. mimicus, and V.fluvialis Phenon 11 comprised 11 strains of unknown identification was effective. However, the scheme which placed reliance which had been isolated from Louisiana and Oregon water on tests for halophilism, carbohydrate fermentation, and samples and could not be identified, even after further other biochemical traits, lacked ability to discriminate ade- phenotypic characterization. The strains were sensitive to quately between V. vulnificus and V. parahaemolyticus. 0/129 (10 Fg), ONPG positive, anaerogenic, arginine Further tests, such as production of acid from arbutin and decarboxylase positive and were variable in the lysine salicin, as well as growth on L-leucine, putrescine, and decarboxylase reaction. We concluded that the cluster rep- ethanol as individual sole carbon sources, need to be in- resents a new species. cluded in the minimum plexus of identification tests, Further Phenon 12, identified as A. hydrophila, included one strain tests suggested for distinguishing these species are listed in provisionally identified as V.fluvialis. Strains of A. hydro- Table 2. Growth on these specific sole carbon sources is phila are frequently isolated from fresh and estuarine waters generally rapid, and the tests can be accomplished in 60 h. but fail to grow well on thiosulfate-citrate-bilesalts-sucrose In summary, there is a clear requirement for the establish- agar. The extensive use of this agar most likely accounts for ment of minimal characteristics for the identification of the few isolates from fresh samples of estuarine and coastal Vibrio spp., especially those isolated from the aquatic envi- waters for study. ronment (15), notably brackish, estuarine, coastal, and oce- Strains of phenon 13 were isolated from Chesapeake Bay anic waters and sediment. New data obtained by 5s ribo- and Puerto Rico seawater and were identified as V.tubiashii. somal ribonucleic acid sequence analysis have confirmed These isolates characteristically degrade tyrosine and xan- these conclusions, especially the validity of the new species thine by means of extracellular enzymes. V. tubiashii was recognized and discussed above (27,28,29). Development of only recently described, and the strains available at that time rapid methods for immunological characterization of patho- were isolated from bacterial disease of mollusk larvae (17). genic vibrios, especially V.vulnificus, can prove very useful The isolation of bacteria pathogenic for oyster larvae from (31, 42, 49) in the field, especially if based on a polyphasic water samples collected from a major seafood harvesting taxonomy of the vibrios (30). estuary is noteworthy. The vibrios in phenon 14, closely related phenotypically to V.tubiashii, could not be identified ACKNOWLEDGMENTS and may represent a biovar of V.tubiashii or a new species. Six decarboxylase-negative vibrios formed a cluster con- We thank N. Roberts, R. Siebeling, R. Seidler, M. Hood, and D. stituting phenon 15. These strains resembled V. splendidus Tison for their excellent collaborative efforts, and we also thank those who provided cultures for this study. on the basis of preliminary screening, but after extensive This work was supported in part by grant BSR-84-01297 from the phenotypic analysis the type strain of this species did not National Science Foundation, grant N00014-81-K-0638 from the cluster within the phenon; thus, this group of organisms Office of Naval Research, and grant C6/181/70 from the World remains unidentified. The taxonomic relationship of these Health Organization. strains to other decarboxylase-negative vibrios was de- scribed previously (45). 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