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JOURNAL OF CLINICAL MICROBIOLOGY, May 1987, p. 900-906 Vol. 25, No. 5 0,095-1137/87/050900-07$02.00/0 Copyright « 1987, American Society for Microbiology veronii, a New Ornithine Decarboxylase-Positive Species That May Cause Diarrheat F. W. HICKMAN-BRENNER,l* K. L. MAcDONALD,2 A. G. STEIGERWALT,3 G. R. FANNING,4 DON J. BRENNER,3 AND J. J. FARMER III1 Enteric Bacteriology Section,' Enteric Diseases Branch,2 and Molecular Biology Laboratory, Meningitis and Special Pathogens Branch,3 Division ofBacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, and Division ofBiochemistry, 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 to our laboratory as "possible 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 other Aeromonas species were 45 to 66% related (60°C) to strain 1169-83, but type strains of 27 Vibrio species were only 2 to 6% related. The name is proposed for the highly related group of nine strains formerly known es Enteric Group 77. The type strain is designated as ATCC 35604 (CDC 1169-83). Strains of A. 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 had negative 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 not fermented: 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 other Aeromonas species. The antibiogram ofA. veronii is typical of other Aeromonas strains (resistance to ampicillin and carbenicillin and susceptibility to most other agents). A. veronii strains were isolated from three clinical sources: respiratory secretions of four victims of drowning or near drowning in fresh water (probably not clinically significant); infected wounds of two patients previously exposed to fresh water (unknown clinical significance); and stools from three patients with (probably clinically significant).

In 1983 we began receiving ornithine decarboxylase- sobria (see Discussion). We instead use the term Aeromonas positive (ornithine+) cultures that resembled hydrophila group to include all the motile species of Aero- biochemically except that they produced gas during fermen- monas that grow readily at 35 to 37°C (mesophilic Aeromo- tation and were string test negative (string test-). We coined nas species). the vernacular name Enteric Group 77 for this organism and Bacterial strains. The 11 ornithine+ strains of Enteric hypothesized that it was a new species of Vibrio closely Group 77 that were studied are listed in Table 1. All strains related to V. cholerae and V. mimicus. Over the next year were maintained in semisolid Trypticase soy agar (9) at room we collected 11 strains. The purposes of this study were to temperature (18 to 28°C) and were also quick-frozen in 10% determine by DNA hybridization, phenotype, and antimicro- skim milk and maintained at -70TC. All results are based on bial susceptibility whether Enteric Group 77 is a new species incubation at 36 ±+1°C unless otherwise noted. in the family and to determine its taxonomic Media and biochemical tests. Commercial media were used position. We also evaluated the clinical significance of whenever possible. The biochemical tests (Table 2) were strains from three different human sources: diarrheal feces, done by methods commonly used in enteric bacteriology, wounds, and the respiratory tract. which have been described in detail elsewhere (5-8). The string test was done by suspending the organism in 0.5% MATERIALS AND METHODS sodium deoxycholate and looking for a string of DNA resulting from cell lysis (7). Most strains of V. cholerae are Nomenclature. We use only names that have standing in string test' within a few seconds. Hemolysis was deter- nonmenclature. In this paper we propose a new species, mined on commercial blood agar plates (Trypticase soy agar Aeromonas veronfi, in the genus Aeromonas of the family containing 5% sheep blood; BBL Microbiology Systems, Vibrionaceae. Because of nomenclatural problems and be- Cockeysville, Md.) at 36°C for 2 days. The production of a cause of the heterogeneity of Aeromonas strains, as indi- diffusible brown pigment was determined on Trypticase soy cated by DNA hybridization, we do not report the two agar at 25 and 36°C for 7 days. Brown pigment was also relatively new species of Aeromonas, A. caviae and A. noted on tyrosine clearing medium (36°C for 7 days). API 20E profiles (Analytab Products, Plainview, N.Y.) were * Corresponding author. determined for the Enteric Group 77 strains in accordance t Dedicated to M. Véron and M. Popoff for their pioneering with the instructions of the manufacturer. The seven-digit studies of the genus Aeromonas. profiles that were obtained were checked in the 1982 API 900 VOL. 25, 1987 AEROMONAS VERONII, A NEW SPECIES 901

TABLE 1. Enteric Group 77 strains studied Other Strain Case ATCCno. Locationsender of Source clinical information A. veronji 0964-83 1 35623 Pennsylvania Stool 74-yr-old female with diarrhea 1169-83Ta 2 35604 Michigan Sputum 10-yr-old male, drowning victim 1170-83 3 35605 Michigan Wound 25-yr-old male with double amputation 1305-83 4 35606 Washington Maxillary sinus 28-yr-old male with left maxillary sinus- itis 0140-84 5 35622 Connecticut Stool 87-yr-old female with diarrhea 0265-84 6 Illinois Endotracheal 11-yr-old female, drowning victim tube 0935-84 7 Ohio Foot wound 6-yr-old female with infected laceration of foot 1067-84 8 New Mexico Stool 80-yr-old male with diarrhea 1068-84 9 North Carolina Lung biopsy 50-yr-old male with Other Enteric Group 77 (ornithine+ Aeromonas strains) 1306-83 10 35941 New Zealand Leg wound 14-yr-old male with compound fracture of left femur 0715-84 il 35942 South Carolina Stool 30-yr-old female with diarrhea and food poisoning a Type strain.

Quick Index and the ninth edition (1985) of the API 20E and to type strains of five other Aeromonas species (Table Analytical Profile Index (computer printout). 3). DNA hybridization. DNA relatedness was determined for Taxonomic position of A. veronhl. The characteristics ofA. all 11 strains of Enteric Group 77 listed in Table 1. Unlabeled veronii, the A. hydrophila group, and the two nonhalophilic DNA was isolated and purified by methods described previ- Vibrio species, V. cholerae and V. mimicus, are shown in ously (3, 4). DNA from strain 1169-83 was labeled with 32p Table 4. A. veronii is more like the A. hydrophila group in by nick translation (in vitro) essentially by the method of every characteristic except lysine and ornithine decarbox- Rigby et al. (15) and in accordance with instructions fur- ylases and arginine dihydrolase. This decarboxylase pattern nished with a commercial nick translation reagent kit (cata- (lysine', arginine-, ornithine+) has never been reported for log no. 8160; Bethesda Research Laboratories, Inc., Aeromonas species but is typical for V. cholerae and V. Gaithersburg, Md.). DNA hybridization experiments were mimicus. However, on the basis ofthe complete biochemical done on hydroxyapatite at 60°C and often at 75°C (4). The pattern, DNA hybridization results, and the G+C content, relatedness of labeled DNA from strain 1169-83 was deter- Enteric Group 77 clearly belongs in the genus Aeromonas. mined against unlabeled DNA from the 10 other Enteric Description of Aeromonas veronii. We propose the name Group 77 strains and stock DNA preparations from 69 other Aeromonas veronii for the nine strains that are listed in strains of the Vibrionaceae. These included type strains of Tables 1 and 3. The species name (pronounced vehr-rhoni-ee- Vibrio, , and Aeromonas species with stand- eye) is derived from the surname of M. M. Véron, a French ing in nomenclature (Table 3), the type strain ofPlesiomonas microbiologist who has contributed greatly to our knowledge shigelloides, and laboratory strains of V. cholerae and the A. of the genera Vibrio and Aeromonas (13, 20, 21) and who hydrophila group. Stock DNA preparations from several coined the family name Vibrionaceae in 1965. The species strains of the family were also tested name is treated as a modern (neo) Latin genitive noun against strain 1169-83 (Table 3). meaning "of Véron." The type strain of the species is G+C content of DNA. The guanine-plus-cytosine (G+C) designated as CDC 1169-83 (ATCC 35604). A complete content of strain 1169-83 (Table 4) was determined description of A. veronii is given in Tables 2 through 6. The spectrophotometrically by the thermal denaturation method complete biochemical reactions of A. veronii are given in (11). Table 2. The unique feature of A. veronii compared with Antibiotic susceptibility tests. Antibiotic susceptibility was other Aeromonas strains was its positive reaction for determined on Mueller-Hinton agar by the disk method of ornithine decarboxylase and negative reaction for arginine Bauer et al. (2). The antibiotics and antimicrobial agents and dihydrolase. The A. veronii strains were string test-, pro- the concentrations used are listed in Table 5. duced gas from D-glucose, were resistant to 0/129 (7), and did not require added NaCl for growth. This is a pattern typical ofAeromonas species. A. veronii had an antibiogram typical of other Aeromonas strains: resistance to penicillin, RESULTS ampicillin, and carbenicillin but susceptibility to chloram- phenicol, colistin, gentamicin, and tetracycline (Table 5). DNA hybridization. By DNA hybridization (Table 3), eight Practical identification of A. veronii. When strain 1169-83 other strains of Enteric Group 77 were very highly related to was compared biochemically with all other strains of the strain 1169-83 (ATCC 35604). Labeled DNA from strain Vibrionaceae and Enterobacteriaceae in our computer data 1169-83 was not highly related to DNA of Vibrio species but base, the ones that were the closest matches were V. was more related to DNA of the type strain of A. hydrophila cholerae strains rather than Aeromonas strains. The nine A. 902 HICKMAN-BRENNER ET AL. J. CLIN. MICROBIOL.

TABLE 2. Biochemical reactions of nine A. veronii strains TABLE 2-Continued Cumulative % Reaction for Cumulative % Reaction for on on positive day: type strain. Test positive day: type strain. Test ATCC 35604 ATCC 35604 1 2 7 (1169-83)" 1 2 7 (1169-83)' Indole production 100 6% 0 0 0 - Indole production (1% NaCl)b 100 + 8% 0 0 0 - Methyl red 89 10% O O O - Methyl red (1% NaCI) 100 + 12% 0 O O - Voges-Proskauer 89 + Brown pigment production on: Voges-Proskauer (1% NaCl) 100 + Trypticase soy agar, 25 and 36°C 0 0 0 Citrate (Simmons') 100 100 100 Tyrosine medium, 25°C 0 0 11 +5 (weak) H2S on triple sugar iron o o o Tyrosine medium, 36°C 0 0 0 H2S on peptone iron agar o o o Beta-hemolysis on sheep blood 100 100 + Urea hydrolysis (Christensen) o o O ' -, Negative at end of appropriate incubation period; +, positive at 24 h or Phenylalanine 89 + time of test. The superscripts indicate the day on which the reaction became Lysine (Moeller) 89 100 100 +2 +2 positive. Lysine (Moeller) (1% NaCI) 78 100 100 b The commercial formula was modified to contain 1% NaCI (final concen- Arginine (Moeller) o o o + tration). Arginine (Moeller) (1% NaCI) o o o +2 Eight strains were tested. Ornithine (Moeller) 67 89 100 d ONPG, o-Nitrophenyl-p-D-galactopyranoside. Ornithine (Moeller) (1% NaCI) 100 100 100 +S Motility 78 100 100 Gelatin hydrolysis (22°C) 22 67 89 KCN, growth in 0 33 78 Malonate utilization o o o veronii strains had seven different API 20E profiles (Table 7). D-Glucose Two of the profiles, 5 346 125 (strains 0935-84 and 1068-84) Acid 100 100 100 and 5 347 125 (strain 0265-84), were identified as V. cholerae Gas 100 100 100 profiles. Therefore, it is important to distinguish strains ofA. Acid from: veronii and V. cholerae. In contrast to V. cholerae strains, Adonitol o o o A. veronii strains produced gas during fermentation and L-Arabinose o o o D-Arabitol o o o were string test- (Table 6). The nine A. veronii strains were Cellobiose 78 78 78 beta-hemolytic on sheep blood within 24 h, and colonies Dulcitol o o o were 2 to 3 mm in diameter. The A. veronii strains grew well Erythritol o o o on MacConkey agar as lactose-negative colonies approxi- D-Galactose 89 100 100 mately 2 mm in diameter (24 h). Only one (1170-83) of eight Glycerol 100 100 100 strains tested grew on thiosulfate-citrate-bile salts-sucrose myo-Inositol o o o agar. The growth was light (plating efficiency less than 10-3), Lactose o o il and the colonies were sucrose+. Maltose 100 100 100 Clinical significance of A. veronii strains. A. veronfi was D-Mannitol 100 100 100 D-Mannose 100 100 100 isolated from stools, wounds, and the respiratory tract. Melibiose o o O Three representative case histories are given below. (We a-Methyl-D-glucoside 89 89 89 include all pertinent information including race because of Raffinose o o o possible correlation.) L-Rhamnose o o O (i) Case 2, sputum (isolate 1169-83, ATCC 35604T). A Salicin 100 100 100 10-year-old male was seen in a hospital emergency room D-Sorbitol o o o after a near-drowning incident in a freshwater lake in Mich- Sucrose 100 100 100 igan. He had bilateral pulmonary infiltrates on a chest X ray Trehalose 100 100 100 with he was afebrile and had a D-Xylose o o o compatible aspiration; Esculin hydrolysis 100 100 100 leukocyte count of 2,400/mm3. A transtracheal aspiration Esculin (1% NaCI) 100 100 100 was performed shortly after his arrival in the hospital emer- Mucate, acid from o o o gency room. Cultures of this aspirated fluid yielded an Tartrate (Jordan) 89 100 100 Aeromonas sp. (not further identified), a Staphylococcus Acetate utilization 56 78 89 sp., and a culture later identified as A. veronii. The patient Lipase (corn oil) 78 100 100 died soon after admission without evidence of pulmonary DNase (25°C) 100 100 100 infection. DNase (360C)c 13 75 88 (il) Case 3, wound (isolate 1170-83, ATCC 35605). A NO3-* NO2 100 Oxidase 100 25-year-old male from Michigan was well until both of his ONPGd test 89 89 89 legs were crushed in an accident. He was trapped under a Citrate (Christensen) 89 100 100 tractor chest deep in a freshwater lake for 20 min before Tyrosine clearing 67 89 89 rescue workers were able to free him. He required above- String test o the-knee amputations of both legs and subsequently devel- Sensitivity to 0/129 o oped gas gangrene in the stump of the left leg. Six days after Growth in nutrient broth plus NaCI his accident, he was transferred to a tertiary care center at: where cultures from his left leg wound yielded several 0% 100 100 100 Clostridium species (C. sporogenes, C. glycolicum, and C. 1% 100 100 100 bifermentans), , , and a strain later identified as A. veronii. He recovered after VOL. 25, 1987 AEROMONAS VERONHI, A NEW SPECIES 903

TABLE 3. DNA relatedness of type strain of A. veronii to strains TABLE 4. Classification of A. veronii of Enteric Group 77 and to other strains of the Vibrionaceae and A. V. Enterobacteriaceae Characteristic A hydrophila cholerae Relatedness (%) to la- group group'' beled DNA of A. ieronii G+C content of DNA (mol%) 58-60 57-63 ATCC 35604lTa 47-49 Source of unlabeled DNA Related- Related- % DNA relatedness (60°C) to: ness at Db ness at V. cholerae type strain 4 3-4 67-100 60°C 75°C A. hydrophila type strain 57 100 4-5 A. veronii strains Five other Aeromonas spp. 45-66 47-73 1-4 1067-84 100 0 100 0265-84 100 0 100 Susceptibility to 0/129 - - + 1169-83 (ATCC 35604T) 100 1 100 0935-84 100 0.5 98 String test - - + 0140-84 (ATCC 35622) 97 0.5 94 0964-83 (ATCC 35623) 94 2.5 93 Biochemical tests 1170-83 (ATCC 35605) 91 2.0 88 Lysine decarboxylase + V + 1068-84 83 1.5 81 Arginine dihydrolase - + 1305-83 (ATCC 35606) 74 1.5 75 Ornithine decarboxylase + - + Gas production + V Other Enteric Group 77 strains (orni- thine+) Susceptibility to ampicillin and R R S 1306-83 49 7.5 26 carbenicillin 0715-84 69 3.5 64 a +, Most strains (generally 90% or more) positive; -, most strains (generally 90o or more) negative; V, variable (some strains negative and some A. hydrophila ATCC 7966Tc (9079-79) 57 8.0 NDd positive; generally 11 to 89% positive); S, susceptible; R, resistant. A. hydrophila subsp. anaerogenes ATCC 54 7.5 ND b V. cholerae and V. mimicus. 15467Tc (9081-79) A. caviae ATCC 15468T (9083-79) 45 9.0 ND A. sobria CIP 7433T (9538-76) 66 5.5 56 A. salmonicida ATCC 33658TC (9701-84) 55 9.0 ND blood, and no fecal leukocytes were noted. His leukocyte A. media ATCC 33907 (9072-83) 47 7.5 ND count was 6,100/mm3 with a normal differential. His diarrhea lasted 4 and without V. cholerae ATCC 14035T (9060-79) 4 ND ND days then resolved spontaneously V. mimicus ATCC 33653T (1721-77) 5 ND ND antimicrobial therapy. Culture of a stool specimen obtained ATCC 17749T 3 ND ND 2 days after the onset of diarrhea did not yield Salmonella, (9065-79) Shigella, or Campylobacter spp. but did yield a strain later ATCC 17802T 3 ND ND identified as A. veronii. A convalescent-phase serum speci- (9062-79) men obtained 18 days after the onset of illness had an ATCC 27562T (9107-79) 4 ND ND antibody titer of 1:512 to the O antigen ofA. veronii 1067-84, Type strains of 25 other Vibrio and Pho- 1-6 ND ND which was isolated from his stool. Six control sera from the tobacterium species Centers for Disease Control Serum Bank had a titer of less than one control serum had a titer of Six of ATCC 14029T 8 ND ND 1:16; 1:32. pools (9091-79) control sera (eight individual control sera in each pool) had a titer of less than 1:16, and one pool had a titer of 1:64. A K-12 4 ND ND commercial human immune serum globulin preparation Other members of the 2-8 ND ND (Cutter Biologicals, Berkeley, Calif.) had a titer of 1:32. The Enterobacteriaceae food exposures of the patient for the week before the onset a T, Type strain. of diarrhea are unknown, but according to his physician, he b D, Divergence, expressed to the nearest 0.5% (3). was drinking untreated well water during that time. He had ATCC 7966 and ATCC 33658 are the type strains for both the species and no history of recent travel outside New Mexico. subspecies; ATCC 15467 is the type strain for the subspecies only. Three patients (cases 1, 5, and 8) had A. veronii isolated d ND, Not determined. than three e Includes all Vibrio and Photobacterium species with standing in nomen- from stools; all had watery diarrhea (more loose clature except V. marinus, V. ordalii, V. carchariae, and V. mediterranei. f Includes Edwardsiella tarda, Hafnia alvei, , Koser- ella trabulsii, Morganella morganii, , Pro'idencia rettgeri, TABLE 5. Antimicrobial susceptibility of nine A. veronii strains Rahnella aquatilis, Salmonella typhimurium, and . Antibiotic (amt/disk) % Susceptible Ampicillin (10 Ftg) ...... 0 Carbenicillin (100 F.g) ...... 0 surgical debridement and intravenous treatment with antibi- 89 otics (tobramycin, clindamycin, and penicillin). Cephalothin (30 p.g) ...... Chloramphenicol (30 Fg) ...... 100 (iii) Case 8, stool (isolate 1067-84). An 80-year-old Native Colistin (10 Fg) ...... 100 American (American Indian) male from New Mexico be- Gentamicin (10 F.g) ...... 100 came ill in September 1984 with an acute onset of frequent Kanamycin (30 F.g) ...... 89 episodes of watery diarrhea. He had no underlying gastro- Nalidixic acid (30 F.g) ...... 89 intestinal diseases but was taking antacids because of Penicillin (10 U)...... 0 chronic renal failure. He had no other symptoms associated Streptomycin (10 Fig) ...... 22 Sulfadiazine (250 p.g) ...... 44 with his diarrhea. He was afebrile, and his abdominal 100 examination was normal. His stool was negative for occult Tetracycline (30 F±g) ...... 904 HICKMAN-BRENNER ET AL. J. CLIN. MICROBIOL.

TABLE 6. Differentiation of A.-veronii from Vibrio spp. and only 3.5%. These findings suggest that this strain is close to other Aeromonas spp. A. veronii but has diverged somewhat. In contrast to A. % Positive" veronii, these two strains were not beta-hemolytic but showed slight alpha-hemolysis. All nine strains of A. veronii A. Halo- were from the United States, but strain 1306-83 was from Test A. hvdro- V. V. philio veronii phila cholerae rnimi(lus New Zealand. The DNA divergence of strain 1306-83 may (9)b group (835) (95) Vibrio represent evolutionary divergence associated with geograph- (100) group ical isolation. Further study of additional ornithine+ isolates Na+ required for growth 0 0 0 0 100 may resolve this issue, but for the present we have classified Gas production 100 40 0 0 strains 0715-84 and 1306-83 as Aeromonas species, String test 0 0 99 100 ornithine+. The patient from whom strain 1306-83 was Lysine decarboxylase 100 50 >99 98 isolated (case 10) did not have any evidence of wound Arginine dihydrolase 0 90 0 0 infection, and drainage was serosanguinous (containing both Ornithine decarboxylase 100 0 98 99 serum Isolate 0715-84 was obtained Acid production from: and blood). (case 11) Salicin 100 35 2 0 from the diarrheal stool of a patient with food poisoning. The Cellobiose 80 50 10 0 diarrhea was self-limiting, and the patient was otherwise oa-Methyl-D-glucoside 90 25 0 0 healthy. No other pathogens were isolated. a Percent positive within 2 days at 36°C. b The number of strains tested is shown in parentheses. DISCUSSION In recent years there has been growing confusion concern- stools per day), with a median duration of 6 days (range, 4 to ing the species of the genus Aeromonas. Two basic subdivi- 15 days). The diarrhea was associated with mild abdominal sions of the genus can be defined based on phenotypic cramping in two patients (cases 1 and 5). None had fever, differences and pathogenicity. The first group is nonmotile, and none had blood in the stools. Leukocyte counts were does not grow at 35 to 37°C, and is pathogenic for . This within normal limits for all patients. Salmonella, Shigella, or is the psychrophilic group that is known as Aeromonas Campylobacter spp. were not isolated from routine stool salmonicida. The second group grows at 35 to 37°C, is specimens. All three patients had evidence of decreased usually motile, and has been isolated from human clinical gastric acidity; two of these (cases 1 and 5) had undergone specimens and other sources. This is the mesophilic group partial gastrectomies, and one (case 8) was taking antacids. that is known as "-in a broad None of the three patients was treated with antimicrobial sense," the A. hydrophila group, or the A. hydrophila agents, and all recovered spontaneously. Clinical histories complex. Strains isolated in clinical microbiology laborato- for these patients suggest that A. veronii may cause a ries belong to this group (16). self-limited watery diarrhea in susceptible hosts, particularly Several proposals to subdivide the A. hydrophila group those with decreased gastric acidity. have recently been made. In the eighth edition of Bergey's In two patients, A. veronii was isolated from leg or foot Manual of Determinative Bacteriology, Schubert (18) di- wounds that were clinically infected (presence of infection vided the A. hydrophila group into two species with five based on clinical evidence). In both, an injury occurred subspecies: A. hydrophila subsp. hydrophila, A. hydrophila while the affected area was submerged in fresh water. One subsp. anaerogenes, A. hydrophila subsp. proteolytica, A. patient (case 3) had gas gangrene, and the other (case 7) had punctata subsp. punctata, and A. punctata subsp. caviae. purulent drainage with surrounding cellulitis. A Streptococ- A. hydrophila subsp. proteolytica is a halophilic vibrio that cus sp. and Staphylococcus aureus were also cultured from has been removed from the genus Aeromonas and is now the wound of the case-7 patient. Because recognized patho- classified as Vibrio proteolyticus. However, the other four gens were cultured from wounds of both patients, the role of organisms are still considered part of the A. hydrophila A. veronfi was unclear. group. Four strains of A. veronii were isolated from respiratory Popoff et al. (14) have subdivided the A. hydrophila group tract specimens. Two patients (cases 2 and 6) were freshwa- in a different way. On the basis of DNA hybridization and ter drowning victims. In both patients A. veronii was isolated phenotype, they recognize three species: A. hydrophila (in a from routine cultures of pulmonary secretions; neither pa- more restricted sense), A. sobria, and A. caviae. This tient had clinical evidence of pneumonia. One strain was classification was used in the chapter on Aeromonas written isolated from a patient with chronic maxillary sinusitis (case for Bergey's Manual of Systematic Bacteriology 4), and S. aureus was cultured from the same specimen. The by Popoff clinical histories of the three patients for whom data were (12). available suggest that A. veronfi was not responsible for TABLE 7. API 20E profiles of nine A. veronii strains clinically significant respiratory tract infection. Two ornithine+ Aeromonas strains that are not A. veronii. Profile Identification Strain(s) with Enteric Group 77 was originally defined on the basis of an profile arbitrary biochemical pattern: lysine ', arginine -, 1 147 124 Nonea 1169-83Tb 1 146 125 None 0964-83 ornithine+, oxidase+, gas+, and string test. Of the original 1 346 125 None 1305-83 11 isolates of Enteric Group 77, 9 were highly related by 1 347 125 None 1170-83 DNA hybridization and also by phenotype. However, two 5 147 125 None 0140-84, 1067-84 isolates (cases 10 and 11, Table 1) were less related. Strain 5 346 125 V. cholerae 0935-84, 1068-84 1306-83 does not belong in the species A. veronii. It was only 5 347 125 V. cholerae 0265-84 42% related to the type strain at 60°C and had a divergence a Indicates that the profile is not listed in the API Quick Index or Computer of 8.5%. Strain 0715-84 was more closely related to A. Index. veronfi: 69% at 60°C and 64% at 75°C, with a divergence of b Type strain. VOL. 25, 1987 AEROMONAS VERONII, A NEW SPECIES 905

We have not used this classification for two reasons. The nary evidence suggests that A. veronii may be a new main reason is that there is a nomenclatural problem with the causative agent. However, the role of Aeromonas spp. in name A. caviae, which appears to be illegitimate. A. human diarrhea is still controversial, and isolation from punctata and A. caviae both have standing in nomenclature diarrheal stools does not necessarily mean the organism and according to the International Code ofNomenclature of caused the illness. Interestingly, one of our patients had a (10), are defined by their type strain, which is high antibody titer to the strain isolated from his feces. This ATCC 15468 for both species (19). Since both organisms was the only serum sample available from any of the have the same type strain, they are objective synonyms (10). patients. In the future, acute- and convalescent-phase sera The oldest published name of two objective synonyms is should be tested against isolates of A. veronii to determine called the senior synonym, and the one published later is whether an antibody response is present. Arrangements for called a junior synonym. The Approved Lists of Bacterial this testing can be made by contacting J. J. Farmer; testing Names (19) fixes 1890 as the publication date for A. is available for all Aeromonas and Plesiomonas cultures. punctata. The publication date for A. caviae is at least 46 years later, 1936 (17; M. Scherago, J. Bacteriol. 31:83, 1936). The International Code of Nomenclature of Bacteria (10) ADDENDUM IN PROOF insists that an organism can have only one correct name, the As part of a larger study on Aeromonas spp. (Fanning et oldest name in compliance with all the rules of nomencla- al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1985), we ture, and it is our opinion that it must be A. punctata rather compared the type strain ofA. veronii by DNA hybridization than A. caviae. A "Request for an Opinion" to the Judicial to a large collection of laboratory and reference Aeromonas Commission of the International Committee on Systematic strains. Some of these Aeromonas strains were highly re- Bacteriology is probably needed to resolve this problem. In lated to A. veronii. However, the highly related strains were the meantime, we are not using A. caviae because it appears lysine+, arginine+, and ornithine-. If these data are con- to be an illegitimate name. The second reason we are not firmed, this group of strains could be defined as a biogroup using the three-species classification is that the A. hydro- (lysine+, arginine+, ornithine-) of A. veronii. This problem phila group consists of 9 to 12 DNA hybridization groups will be addressed in a future publication on the A. hydrophila rather than three distinct species. The original paper of group. Popoff et al. (14) has eight or nine different hybridization groups among the three named species. These findings were LITERATURE CITED confirmed by Fanning et al. (G. R. Fanning, F. W. Hickman- 1. Allen, D. A., B. Austin, and R. R. Colwell. 1983. Aeromonas Brenner, J. J. Farmer, III, and D. J. Brenner, Abstr. Annu. media, a new species isolated from river water. Int. J. Syst. Meet. Am. Soc. Microbiol. 1985, C116, p. 319), who found at Bacteriol. 33:599-604. least 10 hybridization groups among the three species. 2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. The previous discussions indicate that there are additional 1966. Antibiotic susceptibility testing by a standardized single Aeromonas species. Our proposal of A. veronii is an attempt disk method. Am. J. Clin. Pathol. 45:493-496. to better 3. Brenner, D. J., G. R. Fanning, K. E. Johnson, R. V. Citarella, define a small part of this genus. Unfortunately, no and S. Falkow. 1969. Polynucleotide sequence relationships phenotypic tests are available that clearly differentiate all among members of Enterobacteriaceae. J. Bacteriol. 98:637- Aeromonas strains which can be differentiated by DNA 650. hybridization. Until simple methods are found, clinical mi- 4. Brenner, D. J., J. J. Farmer III, G. R. Fanning, A. G. crobiology laboratories must decide how to report Aeromo- Steigerwalt, P. Klykken, H. G. Wathen, F. W. Hickman, and nas cultures. W. H. Ewing. 1978. Deoxyribonucleic acid relatedness of There is no ideal way to report Aeromonas strains from Proteus and Providencia species. Int. J. Syst. Bacteriol. clinical specimens, but we believe that the following recom- 28:269-282. mendations offer the best solutions based on the data cur- 5. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae. 3rd ed., p. 337-354. Burgess Publishing rently available. Most clinical laboratories should not try to Co., Minneapolis. make distinctions among clinical Aeromonas strains and 6. Hickman, F. W., and J. J. Farmer III. 1978. Salmonella typhi: should simply report strains that grow at 35 to 37°C as A. identification, antibiograms, serology, and bacteriophage typ- hydrophila group or as Aeromonas species. The addition of ing. Am. J. Med. Technol. 44:1149-1159. the word group indicates that multiple species are involved. 7. Hickman, F. W., J. J. Farmer MI, D. G. Hollis, G. R. Fanning, Alternative terms are A. hydrophila complex, A. hydrophila A. G. Steigerwalt, R. E. Weaver, and D. J. Brenner. 1982. (broad sense), and A. hydrophila sensu lato, which means Identification of Vibrio hollisae sp. nov. from patients with "in a broad sense." This terminology for Aeromonas is diarrhea. J. Clin. Microbiol. 15:395-401. analogous to the term coagulase-negative staphylococcus or 8. Hickman, F. W., J. J. Framer (sic) III, A. G. Steigerwalt, and D. J. Brenner. 1980. Unusual groups of Morganella Staphylococcus epidermidis group, which is now recognized ("Proteus") morganii isolated from clinical specimens: lysine- to be much broader than the single species S. epidermidis. positive and ornithine-negative biogroups. J. Clin. Microbiol. The term A. hydrophila group would include A. caviae-A. 12:88-94. punctata, A. sobria, A. media (1), and A. veronii. We are not 9. Hickman-Brenner, F. W., G. P. Huntley-Carter, G. R. Fanning, optimistic that a better method of classification and reporting D. J. Brenner, and J. J. Farmer m. 1985. Koserella trabulsii, a will become available soon. However, we are testing all of new genus and species of Enterobacteriaceae formerly known the DNA hybridization groups for a large number of pheno- as Enteric Group 45. J. Clin. Microbiol. 21:39-42. typic characteristics. Perhaps these and similar studies in 10. Lapage, S. P., P. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, laboratories H. P. R. Seeliger, and W. A. Clark (ed.). 1975. International other will provide simple tests that can be used code of nomenclature of bacteria. American Society for Micro- for routine identification of all of the Aeromonas species. biology, Washington, D.C. In this paper we show that A. veronii is a distinct new 11. Marmur, J., and P. Doty. 1962. Determination of the base species. Further studies are needed to better define its composition of deoxyribonucleic acid from its thermal denatur- clinical significance. This is particularly important when the ation temperature. J. Mol. Biol. 5:109-118. isolate is from a patient with diarrhea because our prelimi- 12. Popoff, M. 1984. Genus III. Aeromonas Kluyver and Van Niel 906 HICKMAN-BRENNER ET AL. J. CLIN. MICROBIOL.

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