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Biochemical and exoenzymatic properties of Aeromonas species
DIAGNMICROBIOLINFECTDIS 1985;3:223-232
223
Biochemical and Exoenzymatic Properties of A e r o m o n a s Species J. Michael Janda
One hundred twenty-seven isolates of Aeromonas comprising the three currently recognizable species (A. hydrophila~ A. sobria, and A. caviae) were evaluated for biochemical and exoenzymatic properties. Aeromonas species were generally (>90%) characterized as gram-negative fermentative rods that were oxidase-, catalase-, and ~galactosidase-positive, produced arginine dihydrolase, and failed to decarboxylate ornithine. More than 95% of all isolates tested failed to grow on 6.5% salt or thiosulfate-citrate bile salts agnr and were resistant to the vibriostatic agent 0/129. Most Aeromonas species produced acid from hexoses while failing to ferment alcoholic sugars or trisaccharides. In exoenzymatic studies, Aeromonas species were uniformly found to produce several exoenzymes, including amylase, DNase, RNase, esterase, lipase, gelatinase, protease, fibrinolysin, and chitinase. Within the genus, a number of biochemical and enzymatic properties were found to be associated with one or more of the taxonomically recognizable species. These properties included g/ycoside utilization, Heiberg grouping based upon fermentation of arabinose, sucrose, and mannose, and the elaboration of several extracellular enzymes (elastase, hemolysin, lecithinase, phosphatase). In addition, phenotypic markers previously associated with enteratoxigenie Aeromonas isolates were almost exclusively found among _A.hydrophila and A. sobria species, suggesting that these species are the major enteric pathogens. INTRODUCTION Over the past 20 years, the taxonomic status of species within the genus Aeromonas has been in state of flux. Motile aeromonads have often simply been referred to as the A. hydrophila complex. Recent studies, however, primarily based u p o n numerical taxonomy (Popoff and V6ron, 1976) and polynucleotide sequence relatedness (Popoff et al., 1981) suggest that at least three species of motile aeromonads, namely A. hydrophila, A. sobria, and A. coviae, should be recognized. Within each species, D N A - D N A reassociation kinetics indicate that more than one hybridization group exists, suggesting that further species of biovarieties may" subsequently be delineated. In 1976, Popoff and V6ron (1976) described a number of phenotypic properties useful in the separation of the three Aeromonas species. A previous report from this laboratory showed a distinct association of nine additional properties with one or more of these species. Utilization of these combined properties allowed for speciation of greater than 98% of all clinical isolates (Janda et al., 1984). Researchers investigating the A. hydrophila complex have recently established associations between different biotypes of A. hydrophila and enterotoxigenicity, cytotoxicity, and enhanced virulence for mice (Cumberbatch et al., 1979; Daily et al.,
From the Department of Microbiology, The Mt. Sinai Hospital, New York, New York. Address reprint requests to: J. Michael Janda, Ph.D., Department of Microbiology, Atran 521, The Mount Sinai Hospital, 5th Avenue at 100th Street, New York, NY 10029. Received May 22, 1984: revised and accepted August 27, 1984. Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York. NY 10017 © 1985 Elsevier
0732-8893/85/$03.30
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1981; Burke et al., 1982; Turnbull et al., 1984). Speciation of Aeromonas isolates has additionally shown a correlation between species and virulence potential (Daily et al., 1981), production of cholera-like illness (Champsaur et al., 1982) and specific disease-associated syndromes (Janda et al., 1984). Early studies on the biochemical characterization of the Aeromonas group however, made use of older taxonomy (Ewing et al., 1961; yon Graevenitz, 1980). Therefore, failure to speciate members of the complex has resulted in omission of important phenotypic properties associated with an isolate recovered from a particular clinical syndrome, such as lysine decarboxylase activity with enterotoxigenicity. In this regard, the present report presents an analysis of the biochemical and enzymatic properties of aeromonads in an attempt to correlate identification of species with phenotypes and potential virulence factors related to pathogenicity.
MATERIALS AND METHODS SIxains, Test Conditions, and Reagents One hundred twenty-seven Aeromonas isolates were studied in this survey and were a subset of strains previously reported (Janda et el., 1984). Of the 127 strains analyzed, 111 were isolated from various geographic areas of the United States. Sixty percent of these isolates originated from the New York City area. An additional 16 isolates were kindly provided by P. Echeverria and were recovered in Thailand from the gastrointestinal contents of symptomatic and asymptomatic children. The number and source of the United States isolates were as follows: A. hydrophila (n = 53): gastrointestinal (11), wounds (14), respiratory tract (9), blood (6), environmental (5), and miscellaneous (8); A. sobria (n = 29): gastrointestinal (10), blood (7), wound (4), environmental (3), and miscellaneous (5); A. caviae (n = 29): gastrointestinal (12), wound (7), blood (2), respiratory tract (2), environmental (1), and miscellaneous (5). Definitive identification of each isolate as A. hydrophila, A. sobria, or A. caviae was accomplished through the speciation scheme of Popoff and V6ron (1978) as recently expanded (Janda et el., 1984). Greater than 90% of all strains identified in this manner generated ideal or near ideal phenotypes. Biochemical tests and exoenzyme plate assay results for each Aeromonas isolate were recorded after 72-hr incubation at 37°C unless otherwise stated. Acid production from carbohydrate utilization was assessed after 5 days' incubation. Tests were controlled by inclusion of known strains with positive and negative reactions. Substrates used for carbohydrate fermentation studies or exoenzyme analysis were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise specified. Biochemical media utilized in these studies were purchased from Difco (Detroit, MI) or BBL Laboratories (Cockeysville, MD), and were prepared according to the manufacturer's instructions. Biochemical Tests Biochemical tests were performed according to established methodologies (Ewing and Davis, 1980; MacFaddin, 1980). Test conditions for arbutin hydrolysis were as previously reported (Janda et el., 1984). Oxidase and catalase activities, indole formation, fermentation reactions on triple sugar iron (TSI) and Kligler's iron agar (KIA) slants, growth on thiosulfate citrate bile salts (TCBS) agar (Eiken Chemical Company, Tokyo, Japan), [3-galactosidase activity, and sensitivity to the vibriostatic agent 0/129 2,4 diamino-6,7 diisopropylpteridine (Davis et el., 1981) were evaluated after 24-hr incubation. Motility was determined by point inoculation of 108 cells/ml into the
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225
center of the chemotactic agar of Craven and Montie (1981) containing 1% tryptone, 0.3% yeast extract, 0.5% NaCI, and 0.3% agar, After 24-hr incubation, strains exhibiting concentric spreading of less than 10 mm in diameter from the point inoculum were judged to be defective in chemotactic ability. Production of [~-galactosidase was determined by release of orthophenol from 0-nitrophenyl-~-D-galactopyranoside (ONPG). Acetylmethylcarbinol production (Voges-Proskauer, VP) and acidification of glucose broth (methyl red, MR) were determined after 2 and 5 days, respectively.
Carbohydrate Utilization Acid production from carbohydrates or carbohydrate-like compounds was determined in the oxidation-fermentation (O-F) medium of Hugh and Leifson (1953). Tested at 1% concentrations were: adonitol, n-amygdalin, L-arabinose, D-cellobiose, dextrin, dulcitol, i-erythritol, D-fructose, D-galactose, 1-O-methyl-a-D-glucopyranoside, D-glucose, glycerol, inulin (Difco), i-inositol, lactose, maltose, D-mannitol, Dmannose, a-methyl-D-mannoside, D-melibiose, D-melezitose, raffinose, L-rhamnose, ribose, salicin, n-sorbitol, L-sorbose, sucrose, D-trehalose and D-xylose. A change in color from green (uninoculated) to yellow (acidification) was considered positive.
Biotypes Associated with Enterotoxigenicity Forty-five Aeromonas isolates of gastrointestinal origin were typed for phenotypic markers (lysine decarboxylase (LDC), VP, citrate utilization) previously associated with enterotoxigenicity. A fourth marker, hemolytic activity, was determined for each isolate by spot inoculation onto trypticase soy agar (TSA) containing 5% sheep red blood cells as previously described (Janda and Bottone, 1981).
Exoenzyme Tests The exoenzymatic activities of 127 Aeromonas isolates were individually determined by spot inoculation onto substrate plate assays. The methodologies utilized to detect bacteriolytic activity against various microbial species, chondroitinase, deoxyribonuclease (DNase), elastase (7 days), esterase (1 day), fibrinolysin (1 day), gelatinase (2 days), lecithinase, lipase, and proteolytic (caseinase) activities have been previously described (Janda and Bottone, 1981; Janda et al., 1984). Hydrolysis of 0.2% human albumin (Fraction V, United States Biochemical Corporation, Cleveland, OH) was detected in trypticase soy agar (TSA) as described by Barnes and Melton (1971). Amylase activity was evaluated on Mueller-Hinton agar after 48-hr incubation (Lee, 1976). Production of ~-cellulase was assayed according to Skinner (1960), using 2% cellulose in both buffered mineral salts and in nutrient agar. Chitinase activity (7 days) was assessed on 0.25% chitin (crab shell) mineral agar over a water agar base as reported by Lingappa and Lockwood (1961). Collagenase (7 days) was detected by incorporation of 0.5% (v/v) Vitrogen 100 (Flow Laboratories, McLean, VA) into TSA (Frank and Gerber, 1981). Keratinase activity (7 days) was detected on TSA containing 0.2% keratin (ICN Nutritional Biochemicals, Cleveland, OH) (O'Brien and Davis, 1982). Phosphatase activity was qualitatively measured on nutrient agar plates containing phenolphthalein diphosphate by exposure of previously incubated culture plates to ammonia vapors (MacFaddin, 1980). Bright pink-red colored colonies indicated phosphatase activity. Ribonuclease (RNase) activity was evaluated by the procedure of Miller et al. (1971) by incorporation of 0.2% Torula yeast RNA into a 2% tryptone, 0.5% NaCI, and 1.5% agar medium. In addition to the above described assays, 64 of the 127 Aeromonas isolates were
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further tested for enzymatic activities against 20 chromogenic substrates in the APIZYM (Analytab Products, Plainview, NY) according to the r e c o m m e n d e d procedures of the manufacturer. Relative activity for all three Aeromonas species against each of the 20 substrates was calculated according to the procedure of Waltman et al. (1982). RESULTS Of the biochemical and exoenzymatic properties analyzed for 127 Aeromonas strains, 13 tests yielded positive results for all isolates studied, and i n c l u d e d fermentative metabolism, production of cytochrome oxidase, catalase, [~-galactosidase, acid formed from fermentation of fructose, galactose, glucose, maltose, m a n n i t o l and trehalose, a n d elaboration of amylase, esterase, and lipase activities. In addition, 20 biochemical tests were u n i f o r m l y negative for all aeromonads and i n c l u d e d assays for malonate utilization, formation of hydrogen sulfide from inoculated KIA or TSI slants, pheny l a l a n i n e deaminase, and ornithine decarboxylase activities, production of phenylproprionic acid, degradation of x a n t h i n e or h y p o x a n t h i n e crystals, sensitivity to 0/ 129, elaboration of acid from adonitol, dulcitol, erythritol, inositol, sorbose, and xylose, bacteriolytic activity against Micrococcus luteus and Bacillus, and formation of ~-cellulase, collagenase, and keratinase. Tests producing variable biochemical results are listed i n Tables 1 and 2.
TABLE 1. Variable Biochemical Properties of Aeromonas spp. Property Indole Methyl red Voges-Proskauer Citrate (Simmons) Acetate Urease H2S from GCF Nitrate KCN Lysine decarboxylase Arginine dihydrolase Esculin hydrolysis Arbutin hydrolysis Clearing of tyrosine Motility (chemotactic) Tartrate (Jordan's) Gas from: Glucose Glycerol Lactose KIA: AJA K/A TSI: A/A K/A Growth on TCBS Growth oil 6.5% NaC1
Total Pos. (%)
A. hydrophila
A. sobria
A. caviae
110 94 90 101 108 1 92 126 101 93 115 92 93 123 114 2
(87) (74) (71) (80) (85) (1) (72) (99) (80) (73) (91) (72) (73) (97) (90) (2)
49 30 56 45 50 0 57 57 58 57 56 58 58 58 54 0
(84) (52) (97) (78) (86) -(98) (98) (100) (98) (96) (100) (100) (100) (93) --
33 32 34 25 34 0 35 35 11 35 32 0 1 34 33 2
(94) (91) (97) (71) (97) -(100) (100) (31) (100) (91) -(3) (97) (94) (6)
28 32 0 31 24 1 0 34 32 1 27 34 34 31 27 0
(82) (94) -(91) (71) (3) -(100) (94) (3) (79) (100) (100) (91) (79) --
91 33 16
(72) (26) (13)
57 19 15
(98) (33) (26)
34 14 1
(97) (40) (3)
0 0 0
----
31 96
(24) (76)
13 45
(22) (78)
2 33
(6) (94)
16 18
(47) (53)
95 32 5 4
(75) (25) (4) (3)
38 20 2 1
(66) (34) (3) (2)
24 11 0 0
(69) (31) ---
33 1 3 3
(97) (3) (9) (9)
A e r o m o n a s P h e n o t y p i c Properties
227
More than 90% of the isolates tested were able to clear tyrosine agar; a p p r o x i m a t e l y half of these strains p r o d u c e d a melanin-like (brown) pigment on this m e d i u m . Most strains failed to grow on 6.5% NaC1 agar and on TCBS after overnight incubation. Test results for p r o d u c t i o n of acetylmethylcarbinol, methyl red, H2S formation in GCF m e d i u m , growth in KCN broth, lysine decarboxylase activity, h y d r o l y s i s of esculin and arbutin, and p r o d u c t i o n of gas from carbohydrate fermentation a p p e a r e d to be species-associated. Of the 30 carbohydrate or carbohydrate-like c o m p o u n d s tested, one or more Aerom o n a s strains were able to p r o d u c e acid from 80% of the sugars in O - F media. Production of acid from dextrin, ribose, and sucrose was observed in greater than 90% of all aeromonads studied. Alcoholic sugars were rarely attacked. After 5 days' incubation, 7% of all Aeromonas isolates were both lactose- and sucrose-negative. These latter strains were identified as either A. hydrophila or A. sobria. Speciesrelated carbohydrate fermentation was noted with arabinose, cellobiose, 1-O-methylD-glucopyranoside, glycerol, lactose, mannose, and salicin. Ten carbohydrates were rarely utilized, ranging from a low of 1% with inulin to a high of 9% w i t h amygdalin. Biotyping of members of the family Vibrionaceae was originally achieved through the Heiberg classification system (1934) w h i c h makes use of the fermentation of sucrose, arabinose, and m a n n o s e to distinguish eight biotypes. The 127 Aeromonas isolates could be classified into six Heiberg groups as seen in Table 3. No isolate belonged to either group VI or VIII. In each of the three species tested, one Heiberg group p r e d o m i n a t e d ; these were group III (74%) for A. hydrophila, group I (89%) for A. sobria, and group IV (74%) for A. caviae. No other group contained greater than 21% of any given species.
TABLE 2. A c i d Production from Carbohydrate Utilization Aeromonas
Carbohydrate Amygdalin Arabinose Cellobiose Dextrin 1-O-methyl-a-D glucopyranoside Glycerol Inulin Lactose Mannose a-methyl-D-mannoside Melibiose Melizitose Raffinose Rhamnose Ribose Salicin Sorbitol Sucrose
hydrophila
caviae
sobria
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
12 84 49 126 55
(9) (66) (39) (99) (43)
3 51 7 58 46
(5) (88) (12) (100) (79)
0 1 14 35 8
-(3) (24) (100) (23)
9 32 28 33 1
(26) (94) (82) (97) (3)
100 1 47 100 3 8 5 10 6 126 84 10 116
(79) (1) (37) (79) (2) (6) (4) (8) (5) (99) (66) (8) (91)
58 1 20 57 3 3 3 6 6 58 53 8 52
(100) (2) (34) (98) (5) (5) (5) (10) (10) (100) (91) (14) (90)
33 0 4 35 0 2 1 1 0 35 1 0 31
(94) -(11) (100) -(6) (3) (3) -(100) (3) -(89)
9 0 23 8 0 3 1 3 0 33 30 , 2 ,. 33
(26) -(68) (24) -(9) (3) (9) -(97) (88) (0) (97)
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J.M. J a n d a
T A B L E 3. A s s o c i a t i o n of H e i b e r g G r o u p i n g s w i t h A e r o m o n a s spp. Fermentation pattern
No. of positive patterns
Sucrose
Arabinose
Mannose
A. hydrophila
I
+
-
+
8
31
0
II III IV V VI VII VIII Total
+ + + -
+ + + +
+ + + -
0 43 1 0 0 6 0 58
0 0 0 4 0 0 0 35
1 7 25 0 0 1 0 34
Heiberg group
A. Sobria
A. Caviae
U s i n g m i c r o i d e n t i f i c a t i o n s y s t e m s , a n u m b e r of i n v e s t i g a t o r s h a v e a s s o c i a t e d cert a i n b i o t y p e s of Aeromonas w i t h e n t e r o t o x i g e n i c i t y ( C u m b e r b a t c h et el., 1979; B u r k e et al., 1982; T u r n b u l l et al., 1984). W e a s s e s s e d t h e s e b i o t y p e m a r k e r s (LDC, VP, c i t r a t e , h e m o l y s i s ) for 45 g a s t r o i n t e s t i n a l i s o l a t e s of Aeromonas (Table 4). T w e n t y of 45 i s o l a t e s (44%) p o s s e s s e d t h r e e or all f o u r of t h e b i o t y p i c m a r k e r s a s s o c i a t e d w i t h e n t e r o t o x i g e n i c i t y , a n d 1 0 0 % of t h e s e s t r a i n s w e r e i d e n t i f i e d as e i t h e r A. h y drophila or A. sobria. E i g h t y - s e v e n p e r c e n t of t h e A. hydrophila i s o l a t e s a n d 9 3 % of t h e A. sobria s t r a i n s s u r v e y e d h a d at l e a s t t w o of t h e f o u r m a r k e r s , i n c o n t r a s t to t h e A. caviae s t r a i n s , of w h i c h o n l y 2 0 % s h o w e d h e m o l y t i c a c t i v i t y as t h e s i n g l e biochemical marker associated with enterotoxigenicity. M o r e t h a n 9 0 % of all Aeromonas i s o l a t e s p r o d u c e d c h i t i n a s e , D N a s e , RNase, fibrinolysin, gelatinase, and protease, whereas chondroitinase was detected in only t w o of t h e 127 s t r a i n s s c r e e n e d ( T a b l e 5). Elastase, p h o s p h a t a s e , l e c i t h i n a s e , h e m o l y s i n , a n d b a c t e r i o l y t i c a c t i v i t y a g a i n s t Staphylococcus aureus w e r e f o u n d to b e s p e c i e s - d e p e n d e n t . S i x t y - f o u r s e l e c t e d i s o l a t e s of A e r o m o n a s w e r e f u r t h e r i n v e s t i g a t e d b y t h e A P I - Z Y M s y s t e m ( T a b l e 6). O n l y o n e e n z y m e , C8 e s t e r a s e , w a s f o u n d to b e q u a n t i t a t i v e l y p r e s e n t i n s u b s t a n t i a l l y h i g h a m o u n t s i n all t h r e e s p e c i e s . M o s t s t r a i n s ( m o r e t h a n 90%) p r o d u c e d a l k a l i n e a n d a c i d p h o s p h a t a s e s , C4 e s t e r a s e , 6g a l a c t o s i d a s e , a n d N - a c e t y l - g l u c o s a m i d a s e as w e l l . T r y p s i n a n d [3-glucosidase a c t i v ity a p p e a r e d to b e s p e c i e s - r e l a t e d . T A B L E 4. B i o t y p e s of Aeromonas s p p . R e p o r t e d to b e A s s o c i a t e d w i t h E n t e r o t o x i g e n i c i t y Biotypes ° LDC + + + + + 0 0 Total
Distribution
VP CIT HA No. Pos. A. hydrophila A. sobria A. caviae Phenotype + + 0 + 0 0 0
+ 0 0 0 0 0 0
+ + + 0 0 + 0
3 17 4 3 2 4 12
0 10 2 1 1 1 0 45
3 7 2 2 1 0 0 15
0 0 0 0 0 3 12 15
LDC VP HA CIT
Percent Enterotoxin pos. (range) b 85-98 85-98
87-96 55-64
15
°Abbreviations: LDC, lysine decarboxylase; VP, Voges-Proskauer; CIT, citrate utilization; HA, hemolysis on trypticase soy agar containing 5% sheep blood. bOr cytolysin-associated; data derived from Burke et al., 1982; Cumberbatch et al., 1979; Daily et al., 1981; Pitarangsi et al., 1982; Turnbull et al., 1984.
Aeromonas
Phenotypic
229
Properties
T A B L E 5. V a r i a b l e E x o e n z y m a t i c
Properties of Aeromonas
spp. ° Aeromonas
hydrophila
Albuminase Bacteriolytic Activity S. a u r e u s Chitinase Chondroitinase DNase Elastase Fibrinolysin Gelatinase Hemolysin Lecithinase Phosphatase Protease RNase
sobria
caviae
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
108
(85)
53
(91)
31
(89)
24
(71)
34 115 2 126 71 118 126 92 92 100 122 126
(27) (91) (2) (99) (56) (93) (99) (72) (72) (79) (96) (99)
33 57 2 58 54 57 58 55 48 52 58 58
(57) (98) (3) (100) (93) (98) (100) (95) (83) (90) (100) (100)
1 32 0 35 17 34 34 30 32 33 35 35
(3) (91) -(100) (49) (97) (97) (86) (91) (94) (100) (100)
0 26 0 33 0 27 34 7 12 15 29 33
-(76) -(97) -(79) (100) (21) (35) (44) (85) (97)
abased on a total of 127 tested (A. hydrophila, n = 58; A. sobria, n = 35; A. caviae, n = 34).
T A B L E 6. A P I - Z Y M E n z y m a t i c
Properties of Aeromonas A. h y d r o p h i l a
Enzyme Alk. p h o s p h a t a s e C4 Esterase C8 Esterase C14 Esterase Leucine aminopeptidase Valine a m i n o p e p t i d a s e Cystine aminopeptidase Trypsin Chymotrypsin Acid p h o s p h a t a s e Phosphoamidase a-Galactosidase [3-Galactosidase [3-Glucuronidase [3-Glucosidase e-Glucosidase N-acetyl-glucosamidase ,x-Mannosidase (x-Fucosidase
spp. ° A. sobria
A. caviae
No. Pos.
(%)
Rel A c t ?
(%)
Rel. Act.
(%)
Rel. Act.
(%)
64 63 64 37 54 1 0 50 0 62 55 0 63 5 31 3 63 0 0
(100) (98) (100) (58) (84) (2) -(78) -(97) (86) -(98) (8) (48) (5) (98) ---
2.54 1.75 3.91 1.00 1.50 0 0 1.41 0 2.25 0.88 0 2.08 0 1.33 0.13 2.91 0 0
(100) (100) (100) (54) (79) --(86) -(100) (83) -(100) -(79) (13) (100) ---
1.60 1.75 3.40 0.90 1.45 0 0 2.15 0 1.50 0.85 0 1.40 0 0 0 2.90 0 0
(100) (95) (100] (90) (100) --(90) -(100) (85) -(100) ---(100) ---
2.10 1.70 3.30 0.55 1.15 0.05 0 0.60 0 1.00 1.00 0 1.70 0.25 1.40 0 2.90 0 0
(100) (100) (100) (30) (75) (5) -(55) -(90) (95) -(95) (25) (60) -(95) ---
°Number of strains tested (n = 64): A. hydrophila (n = 24), A. sobria {n = 20], A. caviae (n = 20) bRelative activity was defined as the total numerical value of each isolate of each species divided by the total number of strains tested for each species.
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DISCUSSION The taxonomic studies of Popoff and V6ron (1976) and Popoff et al. (1981) have defined the first major subdivisions within the Aeramonas hydrophila complex and delineated three separate species on the basis of 11 biochemical properties related to speciation. Recent studies from our laboratory support this classification scheme and suggest that most of the variable reactions reported for the A. hydrophila group are either positive or negative for each distinct species (Janda et al., 1984). Excluding biochemical tests previously found to be of value in the taxonomic separation of Aeromonas spp. (Popoff and V6ron, 1976), evaluation of tests for lysine decarboxylase, hemolysis, arbutin hydrolysis, and for production of staphylolytic enzyme aid in the separation of these species. Other tests significantly associated with one or more species, such as lactose positivity with A. caviae, are of less diagnostic significance. Since an earlier report made from this laboratory, Turnbull and colleagues (1984) have confirmed the value of the speciation of motile Aeromonas spp. and, in addition, have proposed other tests of potential value. In both of these studies, acetylmethylcarbinol production by A. sobria was found to be more frequent (93 and 88%, respectively) than that previously reported by Popoff and V6ron (1976) for this species (58%). Because no single test is of absolute predictive value for the speciation of Aeromonas, a constellation of test results must be evaluated. Furthermore, the polynucleotide sequence data of Popoff and co-workers (1981) suggests that more than one DNA-DNA hybridization group exists within each of the three currently recognized Aeromonas species. Thus, further species of biotypes may be defined at a subsequent date. Based upon the results of the present investigation, the genus Aeromonas can be primarily defined as fermentative gram-negative rods that are oxidase- and catalasepositive, fail to decarboxylate ornithine, are [3-galactosidase-positive, and ferment glucose, mannitol, and dextrin but do not attack inositol or xylose. Resistance to 0/ 129 and failure to grow in the presence of 6.5% salt separate this genus from the morphologically and biochemically similar vibrios, particularly V. fluvialis and V. furnissii (Lee et al., 1981; Brenner et al., 1983). Utilization of carbohydrate and carbohydrate-like compounds (Table 2) by Aeromonas spp. suggest that aeromonads as a group ferment most hexoses but rarely attack alcoholic sugars or trisaccharides. Glycoside utilization (esculin, arbutin, salicin) is distinctly species-associated, as is the production of acid from the disaccharides cellobiose and lactose. Separation of species based upon Heiberg grouping (Table 3) is also supported by this study. One of the most intriguing aspects emerging from the biochemical studies of Aeromonas has been the correlation of certain phenotypes with enterotoxigenicity. Cumberbatch et al. (1979) found a distinct association between strains of A. hydrophila recovered from cases of diarrhea with cytotoxin production and positive LDC and VP tests. Since this initial report, other investigators have noted a correlation between citrate utilization and hemolytic activity with enterotoxin production in strains isolated from patients with gastroenteritis. In the present study, the results of phenotype testing of aeromonads of gastrointestinal origin for biotypes previously shown to be associated with enterotoxigenicity suggest that A. hydrophila and A. sobria are the major aeromonad enteric pathogens (Table 4). The latter species has recently been associated with fulminant gastroenteritis mimicking cholera (Champsaur et al., 1982). Whether A. caviae is truly not associated with gastroenteritis, or produces diarrhea by some unknown mechanism, analogous to Plesiomonas shigelloides, remains to be determined. Many of the Aeromonas caviae isolates qualitatively lack exoenzymes such as hemolysin, elastase, and lecithinase that have been associated with enhanced virulence in other bacterial species. Recently Daily and colleagues (1981) have correlated
Aeromonas Phenotypic Properties
231
enhanced virulence in mice with A. sobria strains that elaborate a number of exoenzymes, including hemolysin and elastase. It may well be that overall virulence is species-related and associated with the production of certain exoenzymes. Studies in fish showed decreased virulence in leaches challenged with aeromonads that were VP-negative and that phenotypically resembled A. caviae (Kou, 1972; Wakabayashi, 1981). In a survey of Aeromonas bacteremia, Janda et al. (1984) noted a decreased frequency of this species among blood isolates, which supports the concept of attenuated invasiveness by this species. A sampling of additional enzymatic properties of Aeromonas spp. in the APIZYM system reinforces the previous studies of Waltman et al. (1982), suggesting that properties such as [3-glucosidase production or trypsin-like activity may be additional markers related to certain biovarieties or species of Aeromonas. Further studies using larger numbers of strains are needed to corroborate these findings. The results of this survey suggest that by using a limited number of biochemical tests the microbiologist can obtain a large amount of useful information concerning the species and biotype of individual isolates. Speciation of isolates will allow for the differentiation of strains during nosocomial or community-acquired outbreaks, enhance accumulation of epidemiologic data, and provide information regarding potential differences in the clinical disease spectrum of each species. In addition, by determining the hemolytic, LDC, and VP reactions of Aeromonas isolates recovered from cases of gastroenteritis, one can predict with a fairly high degree of accuracy whether an individual strain produces enterotoxin or not, and thus establish the etiologic agent of the illness.
REFERENCES
Barnes EM, Melton W (1971) Extracellular enzymic activity of poultry spoilage bacteria. J Appl Bacteriol 34:599. Brenner DJ, Hickman-Brenner FW, Lee JV, Steigerwalt AG, Fanning GR, Hollis DG, Farmer III JJ, Weaver RE, Joseph SW, Seidler RJ (1983) Vibrio furnissii (formerly aerogenic biogroup of Vibrio fluvialis), a new species isolated from human feces and the environment. J C/in Microbiol 18:816. Burke V, Robinson J, Atkinson HM, Gracey M (1982) Biochemical characteristics of enterotoxigenic Aeromonas spp. J Clin Microbiol 15:48. Champsaur H, Andremont A, Mathiew D, Rottman E, Auzepy P (1982) Cholera-like illness due to Aeromonas sobria. J Infect Dis 145:248. Craven RC, Montie TC (1981) Motility and chemotaxis of three strains of Pseudomonas aeruginosa used for virulence studies. Can J Microbiol 27:458. Cumberbatch N, Gurwith MJ, Lanston C, Sack RB, Brunton JL (1979) Cytotoxic enterotoxin produced by Aeromonas hydrophila: Relationship of toxigenic isolates to diarrheal disease. Infect Immun 23:829. Daily PO, Joseph WS, Coolbaugh JC, Walker RI, Merrell BR, Rollins DM, Seidler RJ, Colwell RR, Lissner CR (1981) Association of Aeromonas sobria with human infection. J Clin Microbiol 13:769. Davis BR, Fanning GR, Madden JM, Steigerwalt AG, Bradford Jr HB, Smith HL, Brenner DJ (1981) Characterization of biochemically atypical Vibrio cholerae strains and designation of a new pathogenic species, Vibrio mimicus. J Clin Microbiol 14:631. Ewing WH, Hugh R, Johnson JG (1961) Studies on the Aeromonas Group. Center for Disease Control, Atlanta, Georgia. Ewing, WH, Davis BR (1980) Media and Tests for Differentiation of Enterobacteriaceae. HEW Pub. No. (CDC) 80-8236, pp. 1. Frank SK, Gerber JD (1981) Hydrolytic enzymes of Moraxella boris. J Clin Microbiol 13:269.
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von Graevenitz A (1980) Aeromonas and Plesiomonas. In Manual of Clinical Microbiology, 3rd ed. Eds., EH Lennette, A Balows, WJ Hausler Jr, JP Truant. Washington D.C.: American Society for Microbiology, pp 220-225. Heiberg B (1934) Des r~actions de fermentation chez les vibrions. C Roy Soc Bial (Paris) 115:984. Hugh R, Leifson E (1953) The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J Bacteriol 66:24. Janda JM, Bottone EJ (1981) Pseudomonas aeruginosa enzyme profiling: Predictor of potential invasiveness and use as an epidemiological tool. J Clin Microbial 14:55. Janda JM, Reitano M, Bottone EJ (1984) Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum. J Clin Microbial 19:44. Kou GH (1972) Studies on the occurrence and biochemical properties of virulent and avirulent strains of freshwater fish pathogen, Aeromonas liquefacieus. J Fish Soc Taiwan 1:8. Lee WH (1976) Use of Mueller-Hinton agar as amylase testing medium. J Clin Microbiol 4:312. Lee JV, Shread P, Furniss AL, Bryant TN (1981) Taxonomy and description of Vibrio fluvialis sp. nov. (synonym group F. vibrios, group EF6). J Appl Bacterial 50:73. Lingappa Y, Lockwood JL (1961) A chitin medium for isolation, growth and maintenance of actinomycetes. Nature 189:158. MacFaddin JF (1980) Biochemical Tests for Identification of Medical Bacteria. Williams and Wilkins Co., Baltimore. Miller III A, Sandine WE, Elliker PR (1971) Extracellular nuclease in the genus Lactobacillus. J Bacterial 108:604. O'Brien M, Davis GHG (1982) Enzymatic profile of Pseudomonas maltophilia. J Clin Microbial 16:417. Pitarangsi C, Echeverria P, Whitmire R, Tirapat C. Formal S, Dammin GJ, Tingtalapong M (1982) Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: Prevalence among individuals with and without diarrhea in Thailand. Infect Immun 35:666. Popoff MY, Coynault C, Kiredjian M, Lemelin M (1981) Polynucleotide sequence relatedness among motile Aeromonas species. Curr Microbiol 5:109. Popoff M, V6ron M (1976) A taxonomic study of the Aeromonas hydrophila--Aeromonas punctata group. I Gen Microbiol 94:11. Skinner FA (1960) The isolation of anaerobic cellulose---Decomposing bacteria from soil. J Gen Microbiol 22:539. Turnbull PCB, Lee JV, Miliotis MD, .de Walle SV, Koornhof HJ, Jeffery L, Bryant TN (1984) Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species. J Clin Microbiol 19:175. Wakabayashi H, Kanai K, Hsu TC, Egusa S (1981) Pathogenic activities of Aeromonas hydrophila biovar hydrophila (Chester) Popoff and V~ron, 1976 to fishes. Fish Pathol 15:319. Waltman II WD, Shotts EB, Hsu TC (1982) Enzymatic characterization of Aeromonas hydrophila complex by the API ZYM system. J Clin Microbiol 16:692.
223
Biochemical and Exoenzymatic Properties of A e r o m o n a s Species J. Michael Janda
One hundred twenty-seven isolates of Aeromonas comprising the three currently recognizable species (A. hydrophila~ A. sobria, and A. caviae) were evaluated for biochemical and exoenzymatic properties. Aeromonas species were generally (>90%) characterized as gram-negative fermentative rods that were oxidase-, catalase-, and ~galactosidase-positive, produced arginine dihydrolase, and failed to decarboxylate ornithine. More than 95% of all isolates tested failed to grow on 6.5% salt or thiosulfate-citrate bile salts agnr and were resistant to the vibriostatic agent 0/129. Most Aeromonas species produced acid from hexoses while failing to ferment alcoholic sugars or trisaccharides. In exoenzymatic studies, Aeromonas species were uniformly found to produce several exoenzymes, including amylase, DNase, RNase, esterase, lipase, gelatinase, protease, fibrinolysin, and chitinase. Within the genus, a number of biochemical and enzymatic properties were found to be associated with one or more of the taxonomically recognizable species. These properties included g/ycoside utilization, Heiberg grouping based upon fermentation of arabinose, sucrose, and mannose, and the elaboration of several extracellular enzymes (elastase, hemolysin, lecithinase, phosphatase). In addition, phenotypic markers previously associated with enteratoxigenie Aeromonas isolates were almost exclusively found among _A.hydrophila and A. sobria species, suggesting that these species are the major enteric pathogens. INTRODUCTION Over the past 20 years, the taxonomic status of species within the genus Aeromonas has been in state of flux. Motile aeromonads have often simply been referred to as the A. hydrophila complex. Recent studies, however, primarily based u p o n numerical taxonomy (Popoff and V6ron, 1976) and polynucleotide sequence relatedness (Popoff et al., 1981) suggest that at least three species of motile aeromonads, namely A. hydrophila, A. sobria, and A. coviae, should be recognized. Within each species, D N A - D N A reassociation kinetics indicate that more than one hybridization group exists, suggesting that further species of biovarieties may" subsequently be delineated. In 1976, Popoff and V6ron (1976) described a number of phenotypic properties useful in the separation of the three Aeromonas species. A previous report from this laboratory showed a distinct association of nine additional properties with one or more of these species. Utilization of these combined properties allowed for speciation of greater than 98% of all clinical isolates (Janda et al., 1984). Researchers investigating the A. hydrophila complex have recently established associations between different biotypes of A. hydrophila and enterotoxigenicity, cytotoxicity, and enhanced virulence for mice (Cumberbatch et al., 1979; Daily et al.,
From the Department of Microbiology, The Mt. Sinai Hospital, New York, New York. Address reprint requests to: J. Michael Janda, Ph.D., Department of Microbiology, Atran 521, The Mount Sinai Hospital, 5th Avenue at 100th Street, New York, NY 10029. Received May 22, 1984: revised and accepted August 27, 1984. Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York. NY 10017 © 1985 Elsevier
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1981; Burke et al., 1982; Turnbull et al., 1984). Speciation of Aeromonas isolates has additionally shown a correlation between species and virulence potential (Daily et al., 1981), production of cholera-like illness (Champsaur et al., 1982) and specific disease-associated syndromes (Janda et al., 1984). Early studies on the biochemical characterization of the Aeromonas group however, made use of older taxonomy (Ewing et al., 1961; yon Graevenitz, 1980). Therefore, failure to speciate members of the complex has resulted in omission of important phenotypic properties associated with an isolate recovered from a particular clinical syndrome, such as lysine decarboxylase activity with enterotoxigenicity. In this regard, the present report presents an analysis of the biochemical and enzymatic properties of aeromonads in an attempt to correlate identification of species with phenotypes and potential virulence factors related to pathogenicity.
MATERIALS AND METHODS SIxains, Test Conditions, and Reagents One hundred twenty-seven Aeromonas isolates were studied in this survey and were a subset of strains previously reported (Janda et el., 1984). Of the 127 strains analyzed, 111 were isolated from various geographic areas of the United States. Sixty percent of these isolates originated from the New York City area. An additional 16 isolates were kindly provided by P. Echeverria and were recovered in Thailand from the gastrointestinal contents of symptomatic and asymptomatic children. The number and source of the United States isolates were as follows: A. hydrophila (n = 53): gastrointestinal (11), wounds (14), respiratory tract (9), blood (6), environmental (5), and miscellaneous (8); A. sobria (n = 29): gastrointestinal (10), blood (7), wound (4), environmental (3), and miscellaneous (5); A. caviae (n = 29): gastrointestinal (12), wound (7), blood (2), respiratory tract (2), environmental (1), and miscellaneous (5). Definitive identification of each isolate as A. hydrophila, A. sobria, or A. caviae was accomplished through the speciation scheme of Popoff and V6ron (1978) as recently expanded (Janda et el., 1984). Greater than 90% of all strains identified in this manner generated ideal or near ideal phenotypes. Biochemical tests and exoenzyme plate assay results for each Aeromonas isolate were recorded after 72-hr incubation at 37°C unless otherwise stated. Acid production from carbohydrate utilization was assessed after 5 days' incubation. Tests were controlled by inclusion of known strains with positive and negative reactions. Substrates used for carbohydrate fermentation studies or exoenzyme analysis were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise specified. Biochemical media utilized in these studies were purchased from Difco (Detroit, MI) or BBL Laboratories (Cockeysville, MD), and were prepared according to the manufacturer's instructions. Biochemical Tests Biochemical tests were performed according to established methodologies (Ewing and Davis, 1980; MacFaddin, 1980). Test conditions for arbutin hydrolysis were as previously reported (Janda et el., 1984). Oxidase and catalase activities, indole formation, fermentation reactions on triple sugar iron (TSI) and Kligler's iron agar (KIA) slants, growth on thiosulfate citrate bile salts (TCBS) agar (Eiken Chemical Company, Tokyo, Japan), [3-galactosidase activity, and sensitivity to the vibriostatic agent 0/129 2,4 diamino-6,7 diisopropylpteridine (Davis et el., 1981) were evaluated after 24-hr incubation. Motility was determined by point inoculation of 108 cells/ml into the
Aeromonas Phenotypic Properties
225
center of the chemotactic agar of Craven and Montie (1981) containing 1% tryptone, 0.3% yeast extract, 0.5% NaCI, and 0.3% agar, After 24-hr incubation, strains exhibiting concentric spreading of less than 10 mm in diameter from the point inoculum were judged to be defective in chemotactic ability. Production of [~-galactosidase was determined by release of orthophenol from 0-nitrophenyl-~-D-galactopyranoside (ONPG). Acetylmethylcarbinol production (Voges-Proskauer, VP) and acidification of glucose broth (methyl red, MR) were determined after 2 and 5 days, respectively.
Carbohydrate Utilization Acid production from carbohydrates or carbohydrate-like compounds was determined in the oxidation-fermentation (O-F) medium of Hugh and Leifson (1953). Tested at 1% concentrations were: adonitol, n-amygdalin, L-arabinose, D-cellobiose, dextrin, dulcitol, i-erythritol, D-fructose, D-galactose, 1-O-methyl-a-D-glucopyranoside, D-glucose, glycerol, inulin (Difco), i-inositol, lactose, maltose, D-mannitol, Dmannose, a-methyl-D-mannoside, D-melibiose, D-melezitose, raffinose, L-rhamnose, ribose, salicin, n-sorbitol, L-sorbose, sucrose, D-trehalose and D-xylose. A change in color from green (uninoculated) to yellow (acidification) was considered positive.
Biotypes Associated with Enterotoxigenicity Forty-five Aeromonas isolates of gastrointestinal origin were typed for phenotypic markers (lysine decarboxylase (LDC), VP, citrate utilization) previously associated with enterotoxigenicity. A fourth marker, hemolytic activity, was determined for each isolate by spot inoculation onto trypticase soy agar (TSA) containing 5% sheep red blood cells as previously described (Janda and Bottone, 1981).
Exoenzyme Tests The exoenzymatic activities of 127 Aeromonas isolates were individually determined by spot inoculation onto substrate plate assays. The methodologies utilized to detect bacteriolytic activity against various microbial species, chondroitinase, deoxyribonuclease (DNase), elastase (7 days), esterase (1 day), fibrinolysin (1 day), gelatinase (2 days), lecithinase, lipase, and proteolytic (caseinase) activities have been previously described (Janda and Bottone, 1981; Janda et al., 1984). Hydrolysis of 0.2% human albumin (Fraction V, United States Biochemical Corporation, Cleveland, OH) was detected in trypticase soy agar (TSA) as described by Barnes and Melton (1971). Amylase activity was evaluated on Mueller-Hinton agar after 48-hr incubation (Lee, 1976). Production of ~-cellulase was assayed according to Skinner (1960), using 2% cellulose in both buffered mineral salts and in nutrient agar. Chitinase activity (7 days) was assessed on 0.25% chitin (crab shell) mineral agar over a water agar base as reported by Lingappa and Lockwood (1961). Collagenase (7 days) was detected by incorporation of 0.5% (v/v) Vitrogen 100 (Flow Laboratories, McLean, VA) into TSA (Frank and Gerber, 1981). Keratinase activity (7 days) was detected on TSA containing 0.2% keratin (ICN Nutritional Biochemicals, Cleveland, OH) (O'Brien and Davis, 1982). Phosphatase activity was qualitatively measured on nutrient agar plates containing phenolphthalein diphosphate by exposure of previously incubated culture plates to ammonia vapors (MacFaddin, 1980). Bright pink-red colored colonies indicated phosphatase activity. Ribonuclease (RNase) activity was evaluated by the procedure of Miller et al. (1971) by incorporation of 0.2% Torula yeast RNA into a 2% tryptone, 0.5% NaCI, and 1.5% agar medium. In addition to the above described assays, 64 of the 127 Aeromonas isolates were
J.M. Janda
226
further tested for enzymatic activities against 20 chromogenic substrates in the APIZYM (Analytab Products, Plainview, NY) according to the r e c o m m e n d e d procedures of the manufacturer. Relative activity for all three Aeromonas species against each of the 20 substrates was calculated according to the procedure of Waltman et al. (1982). RESULTS Of the biochemical and exoenzymatic properties analyzed for 127 Aeromonas strains, 13 tests yielded positive results for all isolates studied, and i n c l u d e d fermentative metabolism, production of cytochrome oxidase, catalase, [~-galactosidase, acid formed from fermentation of fructose, galactose, glucose, maltose, m a n n i t o l and trehalose, a n d elaboration of amylase, esterase, and lipase activities. In addition, 20 biochemical tests were u n i f o r m l y negative for all aeromonads and i n c l u d e d assays for malonate utilization, formation of hydrogen sulfide from inoculated KIA or TSI slants, pheny l a l a n i n e deaminase, and ornithine decarboxylase activities, production of phenylproprionic acid, degradation of x a n t h i n e or h y p o x a n t h i n e crystals, sensitivity to 0/ 129, elaboration of acid from adonitol, dulcitol, erythritol, inositol, sorbose, and xylose, bacteriolytic activity against Micrococcus luteus and Bacillus, and formation of ~-cellulase, collagenase, and keratinase. Tests producing variable biochemical results are listed i n Tables 1 and 2.
TABLE 1. Variable Biochemical Properties of Aeromonas spp. Property Indole Methyl red Voges-Proskauer Citrate (Simmons) Acetate Urease H2S from GCF Nitrate KCN Lysine decarboxylase Arginine dihydrolase Esculin hydrolysis Arbutin hydrolysis Clearing of tyrosine Motility (chemotactic) Tartrate (Jordan's) Gas from: Glucose Glycerol Lactose KIA: AJA K/A TSI: A/A K/A Growth on TCBS Growth oil 6.5% NaC1
Total Pos. (%)
A. hydrophila
A. sobria
A. caviae
110 94 90 101 108 1 92 126 101 93 115 92 93 123 114 2
(87) (74) (71) (80) (85) (1) (72) (99) (80) (73) (91) (72) (73) (97) (90) (2)
49 30 56 45 50 0 57 57 58 57 56 58 58 58 54 0
(84) (52) (97) (78) (86) -(98) (98) (100) (98) (96) (100) (100) (100) (93) --
33 32 34 25 34 0 35 35 11 35 32 0 1 34 33 2
(94) (91) (97) (71) (97) -(100) (100) (31) (100) (91) -(3) (97) (94) (6)
28 32 0 31 24 1 0 34 32 1 27 34 34 31 27 0
(82) (94) -(91) (71) (3) -(100) (94) (3) (79) (100) (100) (91) (79) --
91 33 16
(72) (26) (13)
57 19 15
(98) (33) (26)
34 14 1
(97) (40) (3)
0 0 0
----
31 96
(24) (76)
13 45
(22) (78)
2 33
(6) (94)
16 18
(47) (53)
95 32 5 4
(75) (25) (4) (3)
38 20 2 1
(66) (34) (3) (2)
24 11 0 0
(69) (31) ---
33 1 3 3
(97) (3) (9) (9)
A e r o m o n a s P h e n o t y p i c Properties
227
More than 90% of the isolates tested were able to clear tyrosine agar; a p p r o x i m a t e l y half of these strains p r o d u c e d a melanin-like (brown) pigment on this m e d i u m . Most strains failed to grow on 6.5% NaC1 agar and on TCBS after overnight incubation. Test results for p r o d u c t i o n of acetylmethylcarbinol, methyl red, H2S formation in GCF m e d i u m , growth in KCN broth, lysine decarboxylase activity, h y d r o l y s i s of esculin and arbutin, and p r o d u c t i o n of gas from carbohydrate fermentation a p p e a r e d to be species-associated. Of the 30 carbohydrate or carbohydrate-like c o m p o u n d s tested, one or more Aerom o n a s strains were able to p r o d u c e acid from 80% of the sugars in O - F media. Production of acid from dextrin, ribose, and sucrose was observed in greater than 90% of all aeromonads studied. Alcoholic sugars were rarely attacked. After 5 days' incubation, 7% of all Aeromonas isolates were both lactose- and sucrose-negative. These latter strains were identified as either A. hydrophila or A. sobria. Speciesrelated carbohydrate fermentation was noted with arabinose, cellobiose, 1-O-methylD-glucopyranoside, glycerol, lactose, mannose, and salicin. Ten carbohydrates were rarely utilized, ranging from a low of 1% with inulin to a high of 9% w i t h amygdalin. Biotyping of members of the family Vibrionaceae was originally achieved through the Heiberg classification system (1934) w h i c h makes use of the fermentation of sucrose, arabinose, and m a n n o s e to distinguish eight biotypes. The 127 Aeromonas isolates could be classified into six Heiberg groups as seen in Table 3. No isolate belonged to either group VI or VIII. In each of the three species tested, one Heiberg group p r e d o m i n a t e d ; these were group III (74%) for A. hydrophila, group I (89%) for A. sobria, and group IV (74%) for A. caviae. No other group contained greater than 21% of any given species.
TABLE 2. A c i d Production from Carbohydrate Utilization Aeromonas
Carbohydrate Amygdalin Arabinose Cellobiose Dextrin 1-O-methyl-a-D glucopyranoside Glycerol Inulin Lactose Mannose a-methyl-D-mannoside Melibiose Melizitose Raffinose Rhamnose Ribose Salicin Sorbitol Sucrose
hydrophila
caviae
sobria
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
12 84 49 126 55
(9) (66) (39) (99) (43)
3 51 7 58 46
(5) (88) (12) (100) (79)
0 1 14 35 8
-(3) (24) (100) (23)
9 32 28 33 1
(26) (94) (82) (97) (3)
100 1 47 100 3 8 5 10 6 126 84 10 116
(79) (1) (37) (79) (2) (6) (4) (8) (5) (99) (66) (8) (91)
58 1 20 57 3 3 3 6 6 58 53 8 52
(100) (2) (34) (98) (5) (5) (5) (10) (10) (100) (91) (14) (90)
33 0 4 35 0 2 1 1 0 35 1 0 31
(94) -(11) (100) -(6) (3) (3) -(100) (3) -(89)
9 0 23 8 0 3 1 3 0 33 30 , 2 ,. 33
(26) -(68) (24) -(9) (3) (9) -(97) (88) (0) (97)
228
J.M. J a n d a
T A B L E 3. A s s o c i a t i o n of H e i b e r g G r o u p i n g s w i t h A e r o m o n a s spp. Fermentation pattern
No. of positive patterns
Sucrose
Arabinose
Mannose
A. hydrophila
I
+
-
+
8
31
0
II III IV V VI VII VIII Total
+ + + -
+ + + +
+ + + -
0 43 1 0 0 6 0 58
0 0 0 4 0 0 0 35
1 7 25 0 0 1 0 34
Heiberg group
A. Sobria
A. Caviae
U s i n g m i c r o i d e n t i f i c a t i o n s y s t e m s , a n u m b e r of i n v e s t i g a t o r s h a v e a s s o c i a t e d cert a i n b i o t y p e s of Aeromonas w i t h e n t e r o t o x i g e n i c i t y ( C u m b e r b a t c h et el., 1979; B u r k e et al., 1982; T u r n b u l l et al., 1984). W e a s s e s s e d t h e s e b i o t y p e m a r k e r s (LDC, VP, c i t r a t e , h e m o l y s i s ) for 45 g a s t r o i n t e s t i n a l i s o l a t e s of Aeromonas (Table 4). T w e n t y of 45 i s o l a t e s (44%) p o s s e s s e d t h r e e or all f o u r of t h e b i o t y p i c m a r k e r s a s s o c i a t e d w i t h e n t e r o t o x i g e n i c i t y , a n d 1 0 0 % of t h e s e s t r a i n s w e r e i d e n t i f i e d as e i t h e r A. h y drophila or A. sobria. E i g h t y - s e v e n p e r c e n t of t h e A. hydrophila i s o l a t e s a n d 9 3 % of t h e A. sobria s t r a i n s s u r v e y e d h a d at l e a s t t w o of t h e f o u r m a r k e r s , i n c o n t r a s t to t h e A. caviae s t r a i n s , of w h i c h o n l y 2 0 % s h o w e d h e m o l y t i c a c t i v i t y as t h e s i n g l e biochemical marker associated with enterotoxigenicity. M o r e t h a n 9 0 % of all Aeromonas i s o l a t e s p r o d u c e d c h i t i n a s e , D N a s e , RNase, fibrinolysin, gelatinase, and protease, whereas chondroitinase was detected in only t w o of t h e 127 s t r a i n s s c r e e n e d ( T a b l e 5). Elastase, p h o s p h a t a s e , l e c i t h i n a s e , h e m o l y s i n , a n d b a c t e r i o l y t i c a c t i v i t y a g a i n s t Staphylococcus aureus w e r e f o u n d to b e s p e c i e s - d e p e n d e n t . S i x t y - f o u r s e l e c t e d i s o l a t e s of A e r o m o n a s w e r e f u r t h e r i n v e s t i g a t e d b y t h e A P I - Z Y M s y s t e m ( T a b l e 6). O n l y o n e e n z y m e , C8 e s t e r a s e , w a s f o u n d to b e q u a n t i t a t i v e l y p r e s e n t i n s u b s t a n t i a l l y h i g h a m o u n t s i n all t h r e e s p e c i e s . M o s t s t r a i n s ( m o r e t h a n 90%) p r o d u c e d a l k a l i n e a n d a c i d p h o s p h a t a s e s , C4 e s t e r a s e , 6g a l a c t o s i d a s e , a n d N - a c e t y l - g l u c o s a m i d a s e as w e l l . T r y p s i n a n d [3-glucosidase a c t i v ity a p p e a r e d to b e s p e c i e s - r e l a t e d . T A B L E 4. B i o t y p e s of Aeromonas s p p . R e p o r t e d to b e A s s o c i a t e d w i t h E n t e r o t o x i g e n i c i t y Biotypes ° LDC + + + + + 0 0 Total
Distribution
VP CIT HA No. Pos. A. hydrophila A. sobria A. caviae Phenotype + + 0 + 0 0 0
+ 0 0 0 0 0 0
+ + + 0 0 + 0
3 17 4 3 2 4 12
0 10 2 1 1 1 0 45
3 7 2 2 1 0 0 15
0 0 0 0 0 3 12 15
LDC VP HA CIT
Percent Enterotoxin pos. (range) b 85-98 85-98
87-96 55-64
15
°Abbreviations: LDC, lysine decarboxylase; VP, Voges-Proskauer; CIT, citrate utilization; HA, hemolysis on trypticase soy agar containing 5% sheep blood. bOr cytolysin-associated; data derived from Burke et al., 1982; Cumberbatch et al., 1979; Daily et al., 1981; Pitarangsi et al., 1982; Turnbull et al., 1984.
Aeromonas
Phenotypic
229
Properties
T A B L E 5. V a r i a b l e E x o e n z y m a t i c
Properties of Aeromonas
spp. ° Aeromonas
hydrophila
Albuminase Bacteriolytic Activity S. a u r e u s Chitinase Chondroitinase DNase Elastase Fibrinolysin Gelatinase Hemolysin Lecithinase Phosphatase Protease RNase
sobria
caviae
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
No. Pos.
(%)
108
(85)
53
(91)
31
(89)
24
(71)
34 115 2 126 71 118 126 92 92 100 122 126
(27) (91) (2) (99) (56) (93) (99) (72) (72) (79) (96) (99)
33 57 2 58 54 57 58 55 48 52 58 58
(57) (98) (3) (100) (93) (98) (100) (95) (83) (90) (100) (100)
1 32 0 35 17 34 34 30 32 33 35 35
(3) (91) -(100) (49) (97) (97) (86) (91) (94) (100) (100)
0 26 0 33 0 27 34 7 12 15 29 33
-(76) -(97) -(79) (100) (21) (35) (44) (85) (97)
abased on a total of 127 tested (A. hydrophila, n = 58; A. sobria, n = 35; A. caviae, n = 34).
T A B L E 6. A P I - Z Y M E n z y m a t i c
Properties of Aeromonas A. h y d r o p h i l a
Enzyme Alk. p h o s p h a t a s e C4 Esterase C8 Esterase C14 Esterase Leucine aminopeptidase Valine a m i n o p e p t i d a s e Cystine aminopeptidase Trypsin Chymotrypsin Acid p h o s p h a t a s e Phosphoamidase a-Galactosidase [3-Galactosidase [3-Glucuronidase [3-Glucosidase e-Glucosidase N-acetyl-glucosamidase ,x-Mannosidase (x-Fucosidase
spp. ° A. sobria
A. caviae
No. Pos.
(%)
Rel A c t ?
(%)
Rel. Act.
(%)
Rel. Act.
(%)
64 63 64 37 54 1 0 50 0 62 55 0 63 5 31 3 63 0 0
(100) (98) (100) (58) (84) (2) -(78) -(97) (86) -(98) (8) (48) (5) (98) ---
2.54 1.75 3.91 1.00 1.50 0 0 1.41 0 2.25 0.88 0 2.08 0 1.33 0.13 2.91 0 0
(100) (100) (100) (54) (79) --(86) -(100) (83) -(100) -(79) (13) (100) ---
1.60 1.75 3.40 0.90 1.45 0 0 2.15 0 1.50 0.85 0 1.40 0 0 0 2.90 0 0
(100) (95) (100] (90) (100) --(90) -(100) (85) -(100) ---(100) ---
2.10 1.70 3.30 0.55 1.15 0.05 0 0.60 0 1.00 1.00 0 1.70 0.25 1.40 0 2.90 0 0
(100) (100) (100) (30) (75) (5) -(55) -(90) (95) -(95) (25) (60) -(95) ---
°Number of strains tested (n = 64): A. hydrophila (n = 24), A. sobria {n = 20], A. caviae (n = 20) bRelative activity was defined as the total numerical value of each isolate of each species divided by the total number of strains tested for each species.
230
J.M. Janda
DISCUSSION The taxonomic studies of Popoff and V6ron (1976) and Popoff et al. (1981) have defined the first major subdivisions within the Aeramonas hydrophila complex and delineated three separate species on the basis of 11 biochemical properties related to speciation. Recent studies from our laboratory support this classification scheme and suggest that most of the variable reactions reported for the A. hydrophila group are either positive or negative for each distinct species (Janda et al., 1984). Excluding biochemical tests previously found to be of value in the taxonomic separation of Aeromonas spp. (Popoff and V6ron, 1976), evaluation of tests for lysine decarboxylase, hemolysis, arbutin hydrolysis, and for production of staphylolytic enzyme aid in the separation of these species. Other tests significantly associated with one or more species, such as lactose positivity with A. caviae, are of less diagnostic significance. Since an earlier report made from this laboratory, Turnbull and colleagues (1984) have confirmed the value of the speciation of motile Aeromonas spp. and, in addition, have proposed other tests of potential value. In both of these studies, acetylmethylcarbinol production by A. sobria was found to be more frequent (93 and 88%, respectively) than that previously reported by Popoff and V6ron (1976) for this species (58%). Because no single test is of absolute predictive value for the speciation of Aeromonas, a constellation of test results must be evaluated. Furthermore, the polynucleotide sequence data of Popoff and co-workers (1981) suggests that more than one DNA-DNA hybridization group exists within each of the three currently recognized Aeromonas species. Thus, further species of biotypes may be defined at a subsequent date. Based upon the results of the present investigation, the genus Aeromonas can be primarily defined as fermentative gram-negative rods that are oxidase- and catalasepositive, fail to decarboxylate ornithine, are [3-galactosidase-positive, and ferment glucose, mannitol, and dextrin but do not attack inositol or xylose. Resistance to 0/ 129 and failure to grow in the presence of 6.5% salt separate this genus from the morphologically and biochemically similar vibrios, particularly V. fluvialis and V. furnissii (Lee et al., 1981; Brenner et al., 1983). Utilization of carbohydrate and carbohydrate-like compounds (Table 2) by Aeromonas spp. suggest that aeromonads as a group ferment most hexoses but rarely attack alcoholic sugars or trisaccharides. Glycoside utilization (esculin, arbutin, salicin) is distinctly species-associated, as is the production of acid from the disaccharides cellobiose and lactose. Separation of species based upon Heiberg grouping (Table 3) is also supported by this study. One of the most intriguing aspects emerging from the biochemical studies of Aeromonas has been the correlation of certain phenotypes with enterotoxigenicity. Cumberbatch et al. (1979) found a distinct association between strains of A. hydrophila recovered from cases of diarrhea with cytotoxin production and positive LDC and VP tests. Since this initial report, other investigators have noted a correlation between citrate utilization and hemolytic activity with enterotoxin production in strains isolated from patients with gastroenteritis. In the present study, the results of phenotype testing of aeromonads of gastrointestinal origin for biotypes previously shown to be associated with enterotoxigenicity suggest that A. hydrophila and A. sobria are the major aeromonad enteric pathogens (Table 4). The latter species has recently been associated with fulminant gastroenteritis mimicking cholera (Champsaur et al., 1982). Whether A. caviae is truly not associated with gastroenteritis, or produces diarrhea by some unknown mechanism, analogous to Plesiomonas shigelloides, remains to be determined. Many of the Aeromonas caviae isolates qualitatively lack exoenzymes such as hemolysin, elastase, and lecithinase that have been associated with enhanced virulence in other bacterial species. Recently Daily and colleagues (1981) have correlated
Aeromonas Phenotypic Properties
231
enhanced virulence in mice with A. sobria strains that elaborate a number of exoenzymes, including hemolysin and elastase. It may well be that overall virulence is species-related and associated with the production of certain exoenzymes. Studies in fish showed decreased virulence in leaches challenged with aeromonads that were VP-negative and that phenotypically resembled A. caviae (Kou, 1972; Wakabayashi, 1981). In a survey of Aeromonas bacteremia, Janda et al. (1984) noted a decreased frequency of this species among blood isolates, which supports the concept of attenuated invasiveness by this species. A sampling of additional enzymatic properties of Aeromonas spp. in the APIZYM system reinforces the previous studies of Waltman et al. (1982), suggesting that properties such as [3-glucosidase production or trypsin-like activity may be additional markers related to certain biovarieties or species of Aeromonas. Further studies using larger numbers of strains are needed to corroborate these findings. The results of this survey suggest that by using a limited number of biochemical tests the microbiologist can obtain a large amount of useful information concerning the species and biotype of individual isolates. Speciation of isolates will allow for the differentiation of strains during nosocomial or community-acquired outbreaks, enhance accumulation of epidemiologic data, and provide information regarding potential differences in the clinical disease spectrum of each species. In addition, by determining the hemolytic, LDC, and VP reactions of Aeromonas isolates recovered from cases of gastroenteritis, one can predict with a fairly high degree of accuracy whether an individual strain produces enterotoxin or not, and thus establish the etiologic agent of the illness.
REFERENCES
Barnes EM, Melton W (1971) Extracellular enzymic activity of poultry spoilage bacteria. J Appl Bacteriol 34:599. Brenner DJ, Hickman-Brenner FW, Lee JV, Steigerwalt AG, Fanning GR, Hollis DG, Farmer III JJ, Weaver RE, Joseph SW, Seidler RJ (1983) Vibrio furnissii (formerly aerogenic biogroup of Vibrio fluvialis), a new species isolated from human feces and the environment. J C/in Microbiol 18:816. Burke V, Robinson J, Atkinson HM, Gracey M (1982) Biochemical characteristics of enterotoxigenic Aeromonas spp. J Clin Microbiol 15:48. Champsaur H, Andremont A, Mathiew D, Rottman E, Auzepy P (1982) Cholera-like illness due to Aeromonas sobria. J Infect Dis 145:248. Craven RC, Montie TC (1981) Motility and chemotaxis of three strains of Pseudomonas aeruginosa used for virulence studies. Can J Microbiol 27:458. Cumberbatch N, Gurwith MJ, Lanston C, Sack RB, Brunton JL (1979) Cytotoxic enterotoxin produced by Aeromonas hydrophila: Relationship of toxigenic isolates to diarrheal disease. Infect Immun 23:829. Daily PO, Joseph WS, Coolbaugh JC, Walker RI, Merrell BR, Rollins DM, Seidler RJ, Colwell RR, Lissner CR (1981) Association of Aeromonas sobria with human infection. J Clin Microbiol 13:769. Davis BR, Fanning GR, Madden JM, Steigerwalt AG, Bradford Jr HB, Smith HL, Brenner DJ (1981) Characterization of biochemically atypical Vibrio cholerae strains and designation of a new pathogenic species, Vibrio mimicus. J Clin Microbiol 14:631. Ewing WH, Hugh R, Johnson JG (1961) Studies on the Aeromonas Group. Center for Disease Control, Atlanta, Georgia. Ewing, WH, Davis BR (1980) Media and Tests for Differentiation of Enterobacteriaceae. HEW Pub. No. (CDC) 80-8236, pp. 1. Frank SK, Gerber JD (1981) Hydrolytic enzymes of Moraxella boris. J Clin Microbiol 13:269.
232
J.M. Janda
von Graevenitz A (1980) Aeromonas and Plesiomonas. In Manual of Clinical Microbiology, 3rd ed. Eds., EH Lennette, A Balows, WJ Hausler Jr, JP Truant. Washington D.C.: American Society for Microbiology, pp 220-225. Heiberg B (1934) Des r~actions de fermentation chez les vibrions. C Roy Soc Bial (Paris) 115:984. Hugh R, Leifson E (1953) The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J Bacteriol 66:24. Janda JM, Bottone EJ (1981) Pseudomonas aeruginosa enzyme profiling: Predictor of potential invasiveness and use as an epidemiological tool. J Clin Microbial 14:55. Janda JM, Reitano M, Bottone EJ (1984) Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum. J Clin Microbial 19:44. Kou GH (1972) Studies on the occurrence and biochemical properties of virulent and avirulent strains of freshwater fish pathogen, Aeromonas liquefacieus. J Fish Soc Taiwan 1:8. Lee WH (1976) Use of Mueller-Hinton agar as amylase testing medium. J Clin Microbiol 4:312. Lee JV, Shread P, Furniss AL, Bryant TN (1981) Taxonomy and description of Vibrio fluvialis sp. nov. (synonym group F. vibrios, group EF6). J Appl Bacterial 50:73. Lingappa Y, Lockwood JL (1961) A chitin medium for isolation, growth and maintenance of actinomycetes. Nature 189:158. MacFaddin JF (1980) Biochemical Tests for Identification of Medical Bacteria. Williams and Wilkins Co., Baltimore. Miller III A, Sandine WE, Elliker PR (1971) Extracellular nuclease in the genus Lactobacillus. J Bacterial 108:604. O'Brien M, Davis GHG (1982) Enzymatic profile of Pseudomonas maltophilia. J Clin Microbial 16:417. Pitarangsi C, Echeverria P, Whitmire R, Tirapat C. Formal S, Dammin GJ, Tingtalapong M (1982) Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: Prevalence among individuals with and without diarrhea in Thailand. Infect Immun 35:666. Popoff MY, Coynault C, Kiredjian M, Lemelin M (1981) Polynucleotide sequence relatedness among motile Aeromonas species. Curr Microbiol 5:109. Popoff M, V6ron M (1976) A taxonomic study of the Aeromonas hydrophila--Aeromonas punctata group. I Gen Microbiol 94:11. Skinner FA (1960) The isolation of anaerobic cellulose---Decomposing bacteria from soil. J Gen Microbiol 22:539. Turnbull PCB, Lee JV, Miliotis MD, .de Walle SV, Koornhof HJ, Jeffery L, Bryant TN (1984) Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species. J Clin Microbiol 19:175. Wakabayashi H, Kanai K, Hsu TC, Egusa S (1981) Pathogenic activities of Aeromonas hydrophila biovar hydrophila (Chester) Popoff and V~ron, 1976 to fishes. Fish Pathol 15:319. Waltman II WD, Shotts EB, Hsu TC (1982) Enzymatic characterization of Aeromonas hydrophila complex by the API ZYM system. J Clin Microbiol 16:692.