JOURNAL OF CLINICAL MICROBIOLOGY, May 1987, p. 900-906 Vol. 25, No. 50,095-1137/87/050900-07$02.00/0Copyright « 1987, American Society for Microbiology

Aeromonas veronii, a New Ornithine Decarboxylase-PositiveSpecies That May Cause Diarrheat

F. W. HICKMAN-BRENNER,l* K. L. MAcDONALD,2 A. G. STEIGERWALT,3 G. R. FANNING,4DON J. BRENNER,3 AND J. J. FARMER III1

Enteric Bacteriology Section,' Enteric Diseases Branch,2 and Molecular Biology Laboratory, Meningitis and SpecialPathogens Branch,3 Division ofBacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta,

Georgia 30333, and Division of Biochemistry, Walter Reed Army Institute ofResearch, Washington, D.C. 203074

Received 12 November 1986/Accepted 5 February 1987

In 1983, the vernacular name Enteric Group 77 was coined for a group of strains that had been referred toour laboratory as "possible Vibrio cholerae except for gas production." By DNA-DNA hybridization(hydroxyapatite, 31P), 8 of 10 strains of Enteric Group 77 were very highly related to the labeled strain 1169-83(74 to 100% at 60°C and 75 to 100% at 75°C; percent divergence, 0.0 to 2.5). Type strains of six otherAeromonas species were 45 to 66% related (60°C) to strain 1169-83, but type strains of 27 Vibrio species wereonly 2 to 6% related. The name Aeromonas veronii is proposed for the highly related group of nine strainsformerly known es Enteric Group 77. The type strain is designated as ATCC 35604 (CDC 1169-83). Strains ofA. veronu grew well at 36°C and had positive reactions at this temperature for indole, methyl red,Voges-Proskauer, citrate, lysine and ornithine decarboxylases, DNase, lipase, and motility; the strains hadnegative reactions for arginine decarboxylase, H2S, urea, and malonate. The following sugars were fermented:D-glucose (acid and gas), cellobiose (seven of nine strains), D-galactose, maltose, D-mannitol, D-mannose,a-methyl-D-glucoside (eight of nine strains), salicin, sucrose, and trehalose. The following sugars were notfermented: adonitol, L-arabinose, D-arabitol, dulcitol, erythritol, myo-inositol, lactose, raffinose, L-rhamnose,D-sorbitol, and D-xylose. The positive ornithine decarboxylase reaction differentiates A. veronii from otherAeromonas species. The antibiogram ofA. veronii is typical of other Aeromonas strains (resistance to ampicillinand carbenicillin and susceptibility to most other agents). A. veronii strains were isolated from three clinicalsources: respiratory secretions of four victims of drowning or near drowning in fresh water (probably notclinically significant); infected wounds of two patients previously exposed to fresh water (unknown clinicalsignificance); and stools from three patients with diarrhea (probably clinically significant).

In 1983 we began receiving ornithine decarboxylase-positive (ornithine+) cultures that resembled Vibrio choleraebiochemically except that they produced gas during fermen-tation and were string test negative (string test-). We coinedthe vernacular name Enteric Group 77 for this organism andhypothesized that it was a new species of Vibrio closelyrelated to V. cholerae and V. mimicus. Over the next yearwe collected 11 strains. The purposes of this study were todetermine by DNA hybridization, phenotype, and antimicro-bial susceptibility whether Enteric Group 77 is a new speciesin the family Vibrionaceae and to determine its taxonomicposition. We also evaluated the clinical significance ofstrains from three different human sources: diarrheal feces,wounds, and the respiratory tract.

MATERIALS AND METHODS

Nomenclature. We use only names that have standing innonmenclature. In this paper we propose a new species,Aeromonas veronfi, in the genus Aeromonas of the familyVibrionaceae. Because of nomenclatural problems and be-cause of the heterogeneity of Aeromonas strains, as indi-cated by DNA hybridization, we do not report the tworelatively new species of Aeromonas, A. caviae and A.

* Corresponding author.t Dedicated to M. Véron and M. Popoff for their pioneering

studies of the genus Aeromonas.

sobria (see Discussion). We instead use the term Aeromonashydrophila group to include all the motile species of Aero-monas that grow readily at 35 to 37°C (mesophilic Aeromo-nas species).

Bacterial strains. The 11 ornithine+ strains of EntericGroup 77 that were studied are listed in Table 1. All strainswere maintained in semisolid Trypticase soy agar (9) at roomtemperature (18 to 28°C) and were also quick-frozen in 10%skim milk and maintained at -70TC. All results are based onincubation at 36 ±+1°C unless otherwise noted.Media and biochemical tests. Commercial media were used

whenever possible. The biochemical tests (Table 2) weredone by methods commonly used in enteric bacteriology,which have been described in detail elsewhere (5-8). Thestring test was done by suspending the organism in 0.5%sodium deoxycholate and looking for a string of DNAresulting from cell lysis (7). Most strains of V. cholerae arestring test' within a few seconds. Hemolysis was deter-mined on commercial blood agar plates (Trypticase soy agarcontaining 5% sheep blood; BBL Microbiology Systems,Cockeysville, Md.) at 36°C for 2 days. The production of adiffusible brown pigment was determined on Trypticase soyagar at 25 and 36°C for 7 days. Brown pigment was alsonoted on tyrosine clearing medium (36°C for 7 days). API20E profiles (Analytab Products, Plainview, N.Y.) weredetermined for the Enteric Group 77 strains in accordancewith the instructions of the manufacturer. The seven-digitprofiles that were obtained were checked in the 1982 API

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AEROMONAS VERONII, A NEW SPECIES 901

TABLE 1. Enteric Group 77 strains studied

Strain Case ATCC Location of Source Other clinical informationno. sender

A. veronji0964-83 1 35623 Pennsylvania Stool 74-yr-old female with diarrhea1169-83Ta 2 35604 Michigan Sputum 10-yr-old male, drowning victim1170-83 3 35605 Michigan Wound 25-yr-old male with double amputation1305-83 4 35606 Washington Maxillary sinus 28-yr-old male with left maxillary sinus-

itis0140-84 5 35622 Connecticut Stool 87-yr-old female with diarrhea0265-84 6 Illinois Endotracheal 11-yr-old female, drowning victim

tube0935-84 7 Ohio Foot wound 6-yr-old female with infected laceration of

foot1067-84 8 New Mexico Stool 80-yr-old male with diarrhea1068-84 9 North Carolina Lung biopsy 50-yr-old male with pneumonia

Other Enteric Group 77(ornithine+ Aeromonasstrains)1306-83 10 35941 New Zealand Leg wound 14-yr-old male with compound fracture of

left femur0715-84 il 35942 South Carolina Stool 30-yr-old female with diarrhea and food

poisoninga Type strain.

Quick Index and the ninth edition (1985) of the API 20EAnalytical Profile Index (computer printout).DNA hybridization. DNA relatedness was determined for

all 11 strains of Enteric Group 77 listed in Table 1. UnlabeledDNA was isolated and purified by methods described previ-ously (3, 4). DNA from strain 1169-83 was labeled with 32pby nick translation (in vitro) essentially by the method ofRigby et al. (15) and in accordance with instructions fur-nished with a commercial nick translation reagent kit (cata-log no. 8160; Bethesda Research Laboratories, Inc.,Gaithersburg, Md.). DNA hybridization experiments weredone on hydroxyapatite at 60°C and often at 75°C (4). Therelatedness of labeled DNA from strain 1169-83 was deter-mined against unlabeled DNA from the 10 other EntericGroup 77 strains and stock DNA preparations from 69 otherstrains of the Vibrionaceae. These included type strains ofVibrio, Photobacterium, and Aeromonas species with stand-ing in nomenclature (Table 3), the type strain ofPlesiomonasshigelloides, and laboratory strains of V. cholerae and the A.hydrophila group. Stock DNA preparations from severalstrains of the family Enterobacteriaceae were also testedagainst strain 1169-83 (Table 3).G+C content of DNA. The guanine-plus-cytosine (G+C)

content of strain 1169-83 (Table 4) was determinedspectrophotometrically by the thermal denaturation method(11).

Antibiotic susceptibility tests. Antibiotic susceptibility wasdetermined on Mueller-Hinton agar by the disk method ofBauer et al. (2). The antibiotics and antimicrobial agents andthe concentrations used are listed in Table 5.

RESULTS

DNA hybridization. By DNA hybridization (Table 3), eightother strains of Enteric Group 77 were very highly related tostrain 1169-83 (ATCC 35604). Labeled DNA from strain1169-83 was not highly related to DNA of Vibrio species butwas more related to DNA of the type strain of A. hydrophila

and to type strains of five other Aeromonas species (Table3).Taxonomic position of A. veronhl. The characteristics of A.

veronii, the A. hydrophila group, and the two nonhalophilicVibrio species, V. cholerae and V. mimicus, are shown inTable 4. A. veronii is more like the A. hydrophila group inevery characteristic except lysine and ornithine decarbox-ylases and arginine dihydrolase. This decarboxylase pattern(lysine', arginine-, ornithine+) has never been reported forAeromonas species but is typical for V. cholerae and V.mimicus. However, on the basis of the complete biochemicalpattern, DNA hybridization results, and the G+C content,Enteric Group 77 clearly belongs in the genus Aeromonas.

Description of Aeromonas veronii. We propose the nameAeromonas veronii for the nine strains that are listed inTables 1 and 3. The species name (pronounced vehr-rhoni-ee-eye) is derived from the surname of M. M. Véron, a Frenchmicrobiologist who has contributed greatly to our knowledgeof the genera Vibrio and Aeromonas (13, 20, 21) and whocoined the family name Vibrionaceae in 1965. The speciesname is treated as a modern (neo) Latin genitive nounmeaning "of Véron." The type strain of the species isdesignated as CDC 1169-83 (ATCC 35604). A completedescription of A. veronii is given in Tables 2 through 6. Thecomplete biochemical reactions of A. veronii are given inTable 2. The unique feature of A. veronii compared withother Aeromonas strains was its positive reaction forornithine decarboxylase and negative reaction for argininedihydrolase. The A. veronii strains were string test-, pro-duced gas from D-glucose, were resistant to 0/129 (7), anddid not require added NaCl for growth. This is a patterntypical ofAeromonas species. A. veronii had an antibiogramtypical of other Aeromonas strains: resistance to penicillin,ampicillin, and carbenicillin but susceptibility to chloram-phenicol, colistin, gentamicin, and tetracycline (Table 5).

Practical identification of A. veronii. When strain 1169-83was compared biochemically with all other strains of theVibrionaceae and Enterobacteriaceae in our computer database, the ones that were the closest matches were V.cholerae strains rather than Aeromonas strains. The nine A.

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902 HICKMAN-BRENNER ET AL.

TABLE 2. Biochemical reactions of nine A. veronii strains

Cumulative % Reaction for

Testpositive on day: type strain.

ATCC 356041 2 7 (1169-83)"

Indole productionIndole production (1% NaCl)bMethyl redMethyl red (1% NaCI)Voges-ProskauerVoges-Proskauer (1% NaCl)Citrate (Simmons')H2S on triple sugar ironH2S on peptone iron agarUrea hydrolysis (Christensen)PhenylalanineLysine (Moeller)Lysine (Moeller) (1% NaCI)Arginine (Moeller)Arginine (Moeller) (1% NaCI)Ornithine (Moeller)Ornithine (Moeller) (1% NaCI)MotilityGelatin hydrolysis (22°C)KCN, growth inMalonate utilizationD-GlucoseAcidGas

Acid from:AdonitolL-ArabinoseD-ArabitolCellobioseDulcitolErythritolD-GalactoseGlycerolmyo-InositolLactoseMaltoseD-MannitolD-MannoseMelibiosea-Methyl-D-glucosideRaffinoseL-RhamnoseSalicinD-SorbitolSucroseTrehaloseD-Xylose

Esculin hydrolysisEsculin (1% NaCI)Mucate, acid fromTartrate (Jordan)Acetate utilizationLipase (corn oil)DNase (25°C)DNase (360C)cNO3-* NO2OxidaseONPGd testCitrate (Christensen)Tyrosine clearingString testSensitivity to 0/129Growth in nutrient broth plus NaCI

at:0%1%

1001008910089100

100 100o oo oo o

8989 10078 100o oo o

67 89100 10078 10022 670 33o o

100 100100 100

o oo oo o

78 78o oo o

89 100100 100

o oo o

100 100100 100100 100

o o89 89o oo o

100 100o o

100 100100 100

o o100 100100 100

o o89 10056 7878 100100 10013 75

10010089 8989 10067 89oo

100ooO

100100

oo

1001001008978o

100100

ooo

78oo

100100

oil

100100100

O89oO

100o

100100

o100100

o1008910010088

8910089

100 100 100100 100 100

+

+++

+2

+2

+

++2

+S

TABLE 2-ContinuedCumulative % Reaction for

Test positive on day: type strain.ATCC 35604

1 2 7 (1169-83)'

6% 0 0 0 -8% 0 0 0 -10% O O O -12% 0 O O -Brown pigment production on:Trypticase soy agar, 25 and 36°C 0 0 0Tyrosine medium, 25°C 0 0 11 +5 (weak)Tyrosine medium, 36°C 0 0 0

Beta-hemolysis on sheep blood 100 100 +' -, Negative at end of appropriate incubation period; +, positive at 24 h or

time of test. The superscripts indicate the day on which the reaction becamepositive.

b The commercial formula was modified to contain 1% NaCI (final concen-tration).

Eight strains were tested.d ONPG, o-Nitrophenyl-p-D-galactopyranoside.

veronii strains had seven different API 20E profiles (Table 7).Two of the profiles, 5 346 125 (strains 0935-84 and 1068-84)and 5 347 125 (strain 0265-84), were identified as V. choleraeprofiles. Therefore, it is important to distinguish strains ofA.veronii and V. cholerae. In contrast to V. cholerae strains,A. veronii strains produced gas during fermentation andwere string test- (Table 6). The nine A. veronii strains werebeta-hemolytic on sheep blood within 24 h, and colonieswere 2 to 3 mm in diameter. The A. veronii strains grew wellon MacConkey agar as lactose-negative colonies approxi-mately 2 mm in diameter (24 h). Only one (1170-83) of eightstrains tested grew on thiosulfate-citrate-bile salts-sucroseagar. The growth was light (plating efficiency less than 10-3),and the colonies were sucrose+.

Clinical significance of A. veronii strains. A. veronfi wasisolated from stools, wounds, and the respiratory tract.Three representative case histories are given below. (Weinclude all pertinent information including race because ofpossible correlation.)

(i) Case 2, sputum (isolate 1169-83, ATCC 35604T). A10-year-old male was seen in a hospital emergency roomafter a near-drowning incident in a freshwater lake in Mich-igan. He had bilateral pulmonary infiltrates on a chest X raycompatible with aspiration; he was afebrile and had aleukocyte count of 2,400/mm3. A transtracheal aspirationwas performed shortly after his arrival in the hospital emer-gency room. Cultures of this aspirated fluid yielded anAeromonas sp. (not further identified), a Staphylococcussp., and a culture later identified as A. veronii. The patientdied soon after admission without evidence of pulmonaryinfection.

(il) Case 3, wound (isolate 1170-83, ATCC 35605). A25-year-old male from Michigan was well until both of hislegs were crushed in an accident. He was trapped under atractor chest deep in a freshwater lake for 20 min beforerescue workers were able to free him. He required above-the-knee amputations of both legs and subsequently devel-oped gas gangrene in the stump of the left leg. Six days afterhis accident, he was transferred to a tertiary care centerwhere cultures from his left leg wound yielded severalClostridium species (C. sporogenes, C. glycolicum, and C.bifermentans), Serratia marcescens, Citrobacter freundii,and a strain later identified as A. veronii. He recovered after

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AEROMONAS VERONHI, A NEW SPECIES 903

TABLE 3. DNA relatedness of type strain of A. veronii to strainsof Enteric Group 77 and to other strains of the Vibrionaceae and

EnterobacteriaceaeRelatedness (%) to la-

beled DNA of A. ieroniiATCC 35604lTa

Source of unlabeled DNARelated- Related-ness at Db ness at60°C 75°C

A. veronii strains1067-84 100 0 1000265-84 100 0 1001169-83 (ATCC 35604T) 100 1 1000935-84 100 0.5 980140-84 (ATCC 35622) 97 0.5 940964-83 (ATCC 35623) 94 2.5 931170-83 (ATCC 35605) 91 2.0 881068-84 83 1.5 811305-83 (ATCC 35606) 74 1.5 75

Other Enteric Group 77 strains (orni-thine+)

1306-83 49 7.5 260715-84 69 3.5 64

A. hydrophila ATCC 7966Tc (9079-79) 57 8.0 NDdA. hydrophila subsp. anaerogenes ATCC 54 7.5 ND

15467Tc (9081-79)A. caviae ATCC 15468T (9083-79) 45 9.0 NDA. sobria CIP 7433T (9538-76) 66 5.5 56A. salmonicida ATCC 33658TC (9701-84) 55 9.0 NDA. media ATCC 33907 (9072-83) 47 7.5 ND

V. cholerae ATCC 14035T (9060-79) 4 ND NDV. mimicus ATCC 33653T (1721-77) 5 ND NDVibrio alginolyticus ATCC 17749T 3 ND ND

(9065-79)Vibrio parahaemolyticus ATCC 17802T 3 ND ND

(9062-79)Vibrio vulnificus ATCC 27562T (9107-79) 4 ND NDType strains of 25 other Vibrio and Pho- 1-6 ND ND

tobacterium species

Plesiomonas shigelloides ATCC 14029T 8 ND ND(9091-79)

Escherichia coli K-12 4 ND NDOther members of the 2-8 ND ND

Enterobacteriaceaea T, Type strain.b D, Divergence, expressed to the nearest 0.5% (3).ATCC 7966 and ATCC 33658 are the type strains for both the species and

subspecies; ATCC 15467 is the type strain for the subspecies only.d ND, Not determined.e Includes all Vibrio and Photobacterium species with standing in nomen-

clature except V. marinus, V. ordalii, V. carchariae, and V. mediterranei.f Includes Edwardsiella tarda, Hafnia alvei, Klebsiella pneumoniae, Koser-

ella trabulsii, Morganella morganii, Proteus mirabilis, Pro'idencia rettgeri,Rahnella aquatilis, Salmonella typhimurium, and Yersinia enterocolitica.

surgical debridement and intravenous treatment with antibi-otics (tobramycin, clindamycin, and penicillin).

(iii) Case 8, stool (isolate 1067-84). An 80-year-old NativeAmerican (American Indian) male from New Mexico be-came ill in September 1984 with an acute onset of frequentepisodes of watery diarrhea. He had no underlying gastro-intestinal diseases but was taking antacids because ofchronic renal failure. He had no other symptoms associatedwith his diarrhea. He was afebrile, and his abdominalexamination was normal. His stool was negative for occult

TABLE 4. Classification of A. veroniiA. V.

Characteristic A hydrophila choleraegroup group''

G+C content of DNA (mol%) 58-60 57-63 47-49

% DNA relatedness (60°C) to:V. cholerae type strain 4 3-4 67-100A. hydrophila type strain 57 100 4-5Five other Aeromonas spp. 45-66 47-73 1-4

Susceptibility to 0/129 - - +

String test - - +

Biochemical testsLysine decarboxylase + V +Arginine dihydrolase - +Ornithine decarboxylase + - +Gas production + V

Susceptibility to ampicillin and R R Scarbenicillina +, Most strains (generally 90% or more) positive; -, most strains

(generally 90o or more) negative; V, variable (some strains negative and somepositive; generally 11 to 89% positive); S, susceptible; R, resistant.

b V. cholerae and V. mimicus.

blood, and no fecal leukocytes were noted. His leukocytecount was 6,100/mm3 with a normal differential. His diarrhealasted 4 days and then resolved spontaneously withoutantimicrobial therapy. Culture of a stool specimen obtained2 days after the onset of diarrhea did not yield Salmonella,Shigella, or Campylobacter spp. but did yield a strain lateridentified as A. veronii. A convalescent-phase serum speci-men obtained 18 days after the onset of illness had anantibody titer of 1:512 to the O antigen ofA. veronii 1067-84,which was isolated from his stool. Six control sera from theCenters for Disease Control Serum Bank had a titer of lessthan 1:16; one control serum had a titer of 1:32. Six pools ofcontrol sera (eight individual control sera in each pool) had atiter of less than 1:16, and one pool had a titer of 1:64. Acommercial human immune serum globulin preparation(Cutter Biologicals, Berkeley, Calif.) had a titer of 1:32. Thefood exposures of the patient for the week before the onsetof diarrhea are unknown, but according to his physician, hewas drinking untreated well water during that time. He hadno history of recent travel outside New Mexico.Three patients (cases 1, 5, and 8) had A. veronii isolated

from stools; all had watery diarrhea (more than three loose

TABLE 5. Antimicrobial susceptibility of nine A. veronii strains

Antibiotic (amt/disk) % SusceptibleAmpicillin (10 Ftg) .............. .................. 0Carbenicillin (100 F.g) .......... ................... 0Cephalothin (30 p.g) ............ ................... 89Chloramphenicol (30 Fg) ........ .................. 100Colistin (10 Fg)................................... 100Gentamicin (10 F.g) ............ ................... 100Kanamycin (30 F.g) ............ ................... 89Nalidixic acid (30 F.g) .......... ................... 89Penicillin (10 U)................................... 0Streptomycin (10 Fig) .......... ................... 22Sulfadiazine (250 p.g).............................. 44Tetracycline (30 F±g) .............................. 100

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904 HICKMAN-BRENNER ET AL.

TABLE 6. Differentiation of A.-veronii from Vibrio spp. andother Aeromonas spp.

% Positive"

A. Halo-Test A. hvdro- V. V. philio

veronii phila cholerae rnimi(lus(9)b group (835) (95) Vibrio

(100) group

Na+ required for growth 0 0 0 0 100Gas production 100 40 0 0String test 0 0 99 100Lysine decarboxylase 100 50 >99 98Arginine dihydrolase 0 90 0 0Ornithine decarboxylase 100 0 98 99Acid production from:

Salicin 100 35 2 0Cellobiose 80 50 10 0oa-Methyl-D-glucoside 90 25 0 0a Percent positive within 2 days at 36°C.b The number of strains tested is shown in parentheses.

stools per day), with a median duration of 6 days (range, 4 to15 days). The diarrhea was associated with mild abdominalcramping in two patients (cases 1 and 5). None had fever,and none had blood in the stools. Leukocyte counts werewithin normal limits for all patients. Salmonella, Shigella, orCampylobacter spp. were not isolated from routine stoolspecimens. All three patients had evidence of decreasedgastric acidity; two of these (cases 1 and 5) had undergonepartial gastrectomies, and one (case 8) was taking antacids.None of the three patients was treated with antimicrobialagents, and all recovered spontaneously. Clinical historiesfor these patients suggest that A. veronii may cause aself-limited watery diarrhea in susceptible hosts, particularlythose with decreased gastric acidity.

In two patients, A. veronii was isolated from leg or footwounds that were clinically infected (presence of infectionbased on clinical evidence). In both, an injury occurredwhile the affected area was submerged in fresh water. Onepatient (case 3) had gas gangrene, and the other (case 7) hadpurulent drainage with surrounding cellulitis. A Streptococ-cus sp. and Staphylococcus aureus were also cultured fromthe wound of the case-7 patient. Because recognized patho-gens were cultured from wounds of both patients, the role ofA. veronfi was unclear.

Four strains of A. veronii were isolated from respiratorytract specimens. Two patients (cases 2 and 6) were freshwa-ter drowning victims. In both patients A. veronii was isolatedfrom routine cultures of pulmonary secretions; neither pa-tient had clinical evidence of pneumonia. One strain wasisolated from a patient with chronic maxillary sinusitis (case4), and S. aureus was cultured from the same specimen. Theclinical histories of the three patients for whom data wereavailable suggest that A. veronfi was not responsible forclinically significant respiratory tract infection.Two ornithine+ Aeromonas strains that are not A. veronii.

Enteric Group 77 was originally defined on the basis of anarbitrary biochemical pattern: lysine ', arginine -,ornithine+, oxidase+, gas+, and string test. Of the original11 isolates of Enteric Group 77, 9 were highly related byDNA hybridization and also by phenotype. However, twoisolates (cases 10 and 11, Table 1) were less related. Strain1306-83 does not belong in the species A. veronii. It was only42% related to the type strain at 60°C and had a divergenceof 8.5%. Strain 0715-84 was more closely related to A.veronfi: 69% at 60°C and 64% at 75°C, with a divergence of

only 3.5%. These findings suggest that this strain is close toA. veronii but has diverged somewhat. In contrast to A.veronii, these two strains were not beta-hemolytic butshowed slight alpha-hemolysis. All nine strains of A. veroniiwere from the United States, but strain 1306-83 was fromNew Zealand. The DNA divergence of strain 1306-83 mayrepresent evolutionary divergence associated with geograph-ical isolation. Further study of additional ornithine+ isolatesmay resolve this issue, but for the present we have classifiedstrains 0715-84 and 1306-83 as Aeromonas species,ornithine+. The patient from whom strain 1306-83 wasisolated (case 10) did not have any evidence of woundinfection, and drainage was serosanguinous (containing bothserum and blood). Isolate 0715-84 (case 11) was obtainedfrom the diarrheal stool of a patient with food poisoning. Thediarrhea was self-limiting, and the patient was otherwisehealthy. No other pathogens were isolated.

DISCUSSION

In recent years there has been growing confusion concern-ing the species of the genus Aeromonas. Two basic subdivi-sions of the genus can be defined based on phenotypicdifferences and pathogenicity. The first group is nonmotile,does not grow at 35 to 37°C, and is pathogenic for fish. Thisis the psychrophilic group that is known as Aeromonassalmonicida. The second group grows at 35 to 37°C, isusually motile, and has been isolated from human clinicalspecimens and other sources. This is the mesophilic groupthat is known as "Aeromonas hydrophila-in a broadsense," the A. hydrophila group, or the A. hydrophilacomplex. Strains isolated in clinical microbiology laborato-ries belong to this group (16).

Several proposals to subdivide the A. hydrophila grouphave recently been made. In the eighth edition of Bergey'sManual of Determinative Bacteriology, Schubert (18) di-vided the A. hydrophila group into two species with fivesubspecies: A. hydrophila subsp. hydrophila, A. hydrophilasubsp. anaerogenes, A. hydrophila subsp. proteolytica, A.punctata subsp. punctata, and A. punctata subsp. caviae.A. hydrophila subsp. proteolytica is a halophilic vibrio thathas been removed from the genus Aeromonas and is nowclassified as Vibrio proteolyticus. However, the other fourorganisms are still considered part of the A. hydrophilagroup.

Popoff et al. (14) have subdivided the A. hydrophila groupin a different way. On the basis of DNA hybridization andphenotype, they recognize three species: A. hydrophila (in amore restricted sense), A. sobria, and A. caviae. Thisclassification was used in the chapter on Aeromonas writtenby Popoff for Bergey's Manual of Systematic Bacteriology(12).

TABLE 7. API 20E profiles of nine A. veronii strains

Profile Identification Strain(s) withprofile

1 147 124 Nonea 1169-83Tb1 146 125 None 0964-831 346 125 None 1305-831 347 125 None 1170-835 147 125 None 0140-84, 1067-845 346 125 V. cholerae 0935-84, 1068-845 347 125 V. cholerae 0265-84

a Indicates that the profile is not listed in the API Quick Index or ComputerIndex.

b Type strain.

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AEROMONAS VERONII, A NEW SPECIES 905

We have not used this classification for two reasons. Themain reason is that there is a nomenclatural problem with thename A. caviae, which appears to be illegitimate. A.punctata and A. caviae both have standing in nomenclatureand according to the International Code ofNomenclature ofBacteria (10), are defined by their type strain, which isATCC 15468 for both species (19). Since both organismshave the same type strain, they are objective synonyms (10).The oldest published name of two objective synonyms iscalled the senior synonym, and the one published later iscalled a junior synonym. The Approved Lists of BacterialNames (19) fixes 1890 as the publication date for A.punctata. The publication date for A. caviae is at least 46years later, 1936 (17; M. Scherago, J. Bacteriol. 31:83, 1936).The International Code of Nomenclature of Bacteria (10)insists that an organism can have only one correct name, theoldest name in compliance with all the rules of nomencla-ture, and it is our opinion that it must be A. punctata ratherthan A. caviae. A "Request for an Opinion" to the JudicialCommission of the International Committee on SystematicBacteriology is probably needed to resolve this problem. Inthe meantime, we are not using A. caviae because it appearsto be an illegitimate name. The second reason we are notusing the three-species classification is that the A. hydro-phila group consists of 9 to 12 DNA hybridization groupsrather than three distinct species. The original paper ofPopoff et al. (14) has eight or nine different hybridizationgroups among the three named species. These findings wereconfirmed by Fanning et al. (G. R. Fanning, F. W. Hickman-Brenner, J. J. Farmer, III, and D. J. Brenner, Abstr. Annu.Meet. Am. Soc. Microbiol. 1985, C116, p. 319), who found atleast 10 hybridization groups among the three species.The previous discussions indicate that there are additional

Aeromonas species. Our proposal of A. veronii is an attemptto better define a small part of this genus. Unfortunately, nophenotypic tests are available that clearly differentiate allAeromonas strains which can be differentiated by DNAhybridization. Until simple methods are found, clinical mi-crobiology laboratories must decide how to report Aeromo-nas cultures.There is no ideal way to report Aeromonas strains from

clinical specimens, but we believe that the following recom-mendations offer the best solutions based on the data cur-rently available. Most clinical laboratories should not try tomake distinctions among clinical Aeromonas strains andshould simply report strains that grow at 35 to 37°C as A.hydrophila group or as Aeromonas species. The addition ofthe word group indicates that multiple species are involved.Alternative terms are A. hydrophila complex, A. hydrophila(broad sense), and A. hydrophila sensu lato, which means"in a broad sense." This terminology for Aeromonas isanalogous to the term coagulase-negative staphylococcus orStaphylococcus epidermidis group, which is now recognizedto be much broader than the single species S. epidermidis.The term A. hydrophila group would include A. caviae-A.punctata, A. sobria, A. media (1), and A. veronii. We are notoptimistic that a better method of classification and reportingwill become available soon. However, we are testing all ofthe DNA hybridization groups for a large number of pheno-typic characteristics. Perhaps these and similar studies inother laboratories will provide simple tests that can be usedfor routine identification of all of the Aeromonas species.

In this paper we show that A. veronii is a distinct newspecies. Further studies are needed to better define itsclinical significance. This is particularly important when theisolate is from a patient with diarrhea because our prelimi-

nary evidence suggests that A. veronii may be a newcausative agent. However, the role of Aeromonas spp. inhuman diarrhea is still controversial, and isolation fromdiarrheal stools does not necessarily mean the organismcaused the illness. Interestingly, one of our patients had ahigh antibody titer to the strain isolated from his feces. Thiswas the only serum sample available from any of thepatients. In the future, acute- and convalescent-phase serashould be tested against isolates of A. veronii to determinewhether an antibody response is present. Arrangements forthis testing can be made by contacting J. J. Farmer; testingis available for all Aeromonas and Plesiomonas cultures.

ADDENDUM IN PROOF

As part of a larger study on Aeromonas spp. (Fanning etal., Abstr. Annu. Meet. Am. Soc. Microbiol. 1985), wecompared the type strain ofA. veronii by DNA hybridizationto a large collection of laboratory and reference Aeromonasstrains. Some of these Aeromonas strains were highly re-lated to A. veronii. However, the highly related strains werelysine+, arginine+, and ornithine-. If these data are con-firmed, this group of strains could be defined as a biogroup(lysine+, arginine+, ornithine-) of A. veronii. This problemwill be addressed in a future publication on the A. hydrophilagroup.

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media, a new species isolated from river water. Int. J. Syst.Bacteriol. 33:599-604.

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ERRATUM

Aeromonas veronii, a New Ornithine Decarboxylase-Positive Species ThatMay Cause Diarrhea

F. W. HICKMAN-BRENNER, K. L. MAcDONALD, A. G. STEIGERWALT, G. R. FANNING, DON J. BRENNER,AND J. J. FARMER III

Enteric Bacteriology Section, Enteric Diseases Branch, and Molecular Biology Laboratory, Meningitis and Special Pathogens Branch,Division of Bacterial Diseases, Center for Infectious Diseases, Centers for Diseuse Control, Atlanta, Georgia 30333, and Division of

Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307

Volume 25, no. 5, p. 900-906. The ATCC numbers for CDC strains 1169-83, 1170-83, and 1305-83 should be 35624 (typestrain), 35625, and 35626, respectively, in Table 1 and throughout the manuscript. (A reprint with these corrections is availablefrom the authors.)

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