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APPumD MICROBIOLOGY, Oct. 1973, p.

550-553 Copyright 0 1973 American Society for Microbiology

Vol. 26, No. 4

Printed in U.SA.

Medium for the Isolation of Aeromonas hydrophila


EMMETT B. SHOITS, JR., AND RICHARD RIMLER College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602

Received for publication 1 June 1973

A new differential medium, Rimler-Shotts, was tested with 109 isolates representing 13 genera of bacteria obtained from aquatic environments and animals. This medium was effective in presumptive identification of the strains of Aeromonas hydrophila examined, with 94% accuracy. Strains of Citrobacter which were hydrogen sulfide-variable could not be separated from A. hydrophila. This medium was designed to facilitate diagnosis of A. hydrophila infections in animals and humans.

The first isolations of microorganisms with characteristics typical for Aeromonas were obtained in the late 1800s. They were associated with diseases of aquatic animals and their environment, but are no longer available for comparison (3, 12, 18, 23). Some 50 years later, additional bacterial types were isolated which were referred to as the C27 group and were later tentatively placed into the genus Aeromonas In 1961, Ewing et al. suggested the adoption of the following species: (i) Aeromonas hydrophila, which includes organisms previously speciated as A. liquefaciens and A. punctata; (ii) A. salmonicida; and (iii) A. shigelloides, previously referred to as the C27 group (6). Since their first description, the motile members of this genus (A. hydrophila and A. shigelloides) have been reported with increasing frequency from disease conditions of both warm- and cold-blooded vertebrates (1, 5, 6, 8, 13, 16, 17). A number of methods and schemes for the isolation and identification of these organisms have been developed, usually requiring several days to a week to complete (5, 6, 8-10). Current interest in fish farming, the ecological impact, and increased awareness in both veterinary and human medicine have brought this group and, in particular, A. hydrophila to the forefront (13, 16, 17). A. hydrophila is a gram-negative fermentative organism belonging to the order Pseudomonodales. It has many biochemical characteristics similar to some genera of the family Enterobacteraceae. Due to these similarities, A. hydrophila is often difficult to recognize, especially from mixed populations of gram-negative bacteria, and on chance isolation

may be mislabeled. It was with these problems in mind that the reported work was done. The purpose of this study was to develop a simple medium with a differential potential for the rapid isolation and identification of A. hydrophila.
METHODS AND MATERIALS A medium referred to as Rimler-Shotts (RS), which is a modification from several media developed for enterobacteria, was prepared (11, 14, 15). It was composed of L-lysine-hydrochloride, 5 g; L-ornithinehydrochloride, 6.5 g; maltose, 3.5 g; sodium thiosulfate, 6.8 g; L-cysteine-hydrochloride, 0.3 g; bromothymol blue, 0.03 g; ferric ammonium citrate, 0.8 g; sodium deoxycholate, 1.0 g; novobiocin, 0.005 g; yeast extract, 3.0 g; sodium chloride, 5.0 g; agar, 13.5 g; and water in sufficient quantity to make 1 liter. Components were dissolved by stirring; pH was adjusted to 7.0; and the mixture was heated to a boil for 1 min, cooled to 45 C, and poured into plates. Prepared plates were refrigerated until needed. Other media used in this study included MacConkey agar, triple sugar iron agar, Trypticase soy broth, maltose broth, Moeller ornithine broth, and Moeller lysine broth. All of these media were obtained from BBL and were prepared and used as recommended. Prior to the inoculation of media, all organisms were grown in Trypticase soy broth and diluted 1:10 in sterile physiological saline. In instances where enumeration was desired, standard procedures were followed in making logarithmic dilutions with sterile physiologic saline (2). Fractions of these dilutions were plated in triplicate. The inoculum was then spread over the surface of the plates with a glass rod. All inoculated plates and tubes were incubated at 37 C and observed, and findings were recorded at 18, 20, and 24 h. A wide variety of organisms was evaluated on RS medium. These organisms represented 109 isolates belonging to genera frequently associated with water

(7).

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VOL. 26, 1973


or

MEDIUM FOR ISOLATION OF A. HYDROPHILA

fecal contamination, or both (Table 1). The identity of all organisms used in this study was verified by standard biochemical characterization (4).

RESULTS It was found that four characteristic types of colonies were obtained when organisms were inoculated on RS medium. The first was yellow, which indicated maltose fermentation. The second was yellow with a black center, which indicated the same type of reaction as the first but with H2S production. The third colonial type was one which produced shades of greenish-yellow to green, which indicated decarboxylation of lysine or ornithine, or both. A fourth type was green with a black center, indicating the same reaction as the third but with H2S production. Growth inhibition occurred from time to time with members of the genus Proteus and A. shigelloides, although inhibitors were added to retard other species. A correlation of
TABLE 1. Organisms used for comparative study of

reactions
Organism

on

Rimler-Shotts medium
No. of strains
20
14

Source

Aeromonas hydrophila

Escherichia coli
Shigella type A
C

Fish, rhino, human, mink, water, bovine Canine, feline, equine, bovine, porcine, racoon
Human Human Human Human, monkey Canine, porcine, hamster Duck, human, porcine, bovine

D B Proteus mirabilis Citrobacter spp.

2 5 7
9
10 10

Klebsiella spp.
Enterobacter spp. Salmonella
spp.

Human, feline, canine, bovine

Canine, porcine, equine,


bovine, caprine

Turkey, chicken, pigeon,


bovine Human Porcine Chicken

typhi cholerasius pullorum Arizona types 1OA-1OB 7 24 5 Herellea vaginicola Pseudomonas spp. Edwardsiella tarda Serratia marcescens P. rettgeri P. morgani P. vulgaris A. shigelloides

Chicken Turkey
Goose Canine Porcine Water, canine, human Snake Bovine Porcine Bovine Bovine Fish

551 colonial types associated with the genera studied is presented in Table 2. Studies to determine the most ideal time for interpretation and further study of organisms grown on RS medium suggest that plates should be observed after the 20th h but no later than the 24th h. Plates observed after the 26th h demonstrated a slow reversion of the fermentative reaction (yellow) toward a basic reaction (green). Only in a minimal number of cases did the medium fail to react as anticipated. Exceptions included: 1 of 20 (5%) A. hydrophila, 1 of 13 (7.5%) Salmonella spp. (a strain of S. typhi which was only weakly lysine positive and ornithine negative), and 5 of 7 (71%) Citrobacter spp. (This was related to the H2S production by the strains of Citrobacter spp. studied. It was found that three of the five strains in question only produced H2S in colonially crowded areas, whereas only about half of the colonies from the remaining two strains produced H2S.) Of the 109 strains of organisms studied, only 7 failed to react as anticipated (Table 3). Two of these cases, an isolate of A. hydrophila and an isolate of S. typhi, could not be considered as characteristic strains. RS medium produced characteristic, anticipated reactions, based on conventional biochemical studies, in all cases except for the H2S-variable reactions encountered with Citrobacter spp. This medium was effective in identifying A. hydrophila without further study in 104 of 109 instances (95%). If all strains, aberrant and otherwise, are considered, the performance of RS medium in detecting A. hydrophila on initial culture and without further study was 94%. The sensitivity of RS medium was studied in a comparison with MacConkey agar. MacConkey agar was chosen for comparison since it is capable of growing most, if not all, of the gram-negative organisms of the enteric and associated groups. No significant difference in the ability to propagate A. hydrophila was observed between these media when seven randomly selected strains of A. hydrophila were

compared.
bovine,

1
1

8 1
1 1

2
4

109

DISCUSSION The ingredients used in this medium were selected and compounded so as to achieve a maximal acidic (maltose fermentation) or basic reaction (decarboxylation of lysine or ornithine, or both). Hydrogen sulfide production was primarily dependent upon the utilization of sodium thiosulfate or L-cysteine-hydrochloride, or both, with ferric ammonium citrate being uti-

552

SHOWPS AND RIMLER

APPL. MICROBIOL.

lized to help visualize this reaction. The inhibitors, sodium deoxycholate and novobiocin, were added to eliminate chance gram-positive organisms and Vibrio spp. which may have caused false reactions. The use of novobiocin to inhibit the growth of Vibrio spp. minimizes the confusion often encountered in differentiation of these organisms from anaerogenic strains of A. hydrophila. The balance of the ingredients provide a nutrient base and chemophysical stability for the medium.
TABLE 2. Colony characteristics of various gram-negative organisms on Rimler-Shotts medium
Characteristic
Yellow colony

It is important that the medium be incubated at 37 C to eliminate the possible growth of A.

Organisms
Aeromonas hydrophilia (Salmonella typhi) Citrobacter spp. (HSI Proteus vulgaris Citrobacter spp. Salmonella spp. Escherichia spp. Klebsiella spp. Enterobacter spp. Proteus spp. Shigella spp. Pseudomonas spp. Aeromonas shigelloides Herellea Citrobacter spp. Salmonella spp. Arizona spp. Edwardsiella spp. Proteus mirabilis Proteus vulgaris (50%) Aeromonas shigelloides (50%)

Yellow colony with black center Greenish yellow to green

Green colony with black center

Complete growth inhibition at 24 h

salmonicida which would occur at reduced temperatures. This organism will produce a characteristic yellow colony on RS medium at reduced temperature. Since color changes occur due to pH reactions, colonial growth must be observed between the 20th and 24th h for maximal accuracy. It is postulated that after this time interaction in the ornithine-arginine metabolic pathways brings about a reversion of pH. Experience indicates that when only yellow colonies are present they are usually A. hydrophila. However, all yellow colonies, especially those interspersed with yellow ones having black centers, should be subsequently tested for oxidase activity to eliminate the possibility of Citrobacter spp. and/or other organisms. It is encouraging to note that within the strains of the genera studied that the maltose and/or lysine and ornithine reactions have been stable for the most part, suggesting that the bulk of false positives to be anticipated will be members of the genus Citrobacter. The sensitivity of this medium renders it as a very useful medium not only for the recovery of A. hydrophila from specific sources but for the enumeration of this organism in the environment. Preliminary work shows promise that the medium can be modified and used with a membrane filter (Millipore Corp.) for enumeration. The distinct colonial types seen on RS medium add further to its usefulness in selection of suspicious colonies of potentially patho-

TABLE 3. Reactions of various organisms on Rimler-Shotts medium in relation to standard biochemical reactions
No. of strains producing yellow colonies/total tested

Organism
18h

RS medium
20h
24 h

Maltose 24h

Lysine
24 h

Ornithine
24h

HS

24h

A. hydrophila E. coli Shigella Klebsiella Enterobacter Salmonella Arizona Edwardsiella Citrobacter Proteus vulgaris Proteus rettgeri Proteus morganii Proteus mirabilis Serratia marcescens A. shigelloides Pseudomonas Herellea

19/20 1/14 0/5 2/9 1/10 1/13 0/7 0/1 5/7 1/2 0/1 0/1

19/20 0/14 0/5 1/9 0/10 1/13 0/7 0/1 5/7 1/2 0/1 0/1

19/20 0/14 0/5 0/9 0/10 1/13 0/7 0/1 5/7 0/2 0/1 0/1

0/5 0/1 0/4 0/8 0/1

0/5 0/1 0/4


0/8 0/1

0/5 0/1 0/4


0/8 0/1

20/20 14/14 1/5 9/9 10/10 12/13 7/7 1/1 7/7 2/2 0/1 0/1 0/5 1/1 4/4 0/8 0/1

1/20 14/14 0/5

9/9 3/10 13/13 7/7 0/1 0/7 0/2 0/1 0/1 0/5 1/1 4/4 0/8 0/1

0/20 12/14 1/5 0/9

0/20 0/14
0/5 0/9 0/10 13/13 7/7 1/1 7/7 2/2 0/1

10/10
12/13 7/7 1/1 1/7

0/2 0/1 1/1 3/5 0/1 4/4 0/8 0/1

0/1 5/5 0/1 0/4


0/8 0/1

VOL. 26, 1973

ON OF A. HYDROPHILA MEDIUM FOR ISOLATIff


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genic members of the Enterobacteraceae for inoculation to differential media, and in subsequent identification. This medium has been in routine use in the diagnostic laboratories at the College of Veterinary Medicine, University of Georgia, for the past year. During this time, the isolations of A. hydrophila have increased approximately 10fold. The facility with which the medium can be prepared and used without autoclaving permits wide spread use in epidemiological, ecological, and epizootological investigations, as well as routine use in the rapid isolation and identification of A. hydrophila from aquatic and domestic animals, wildlife, and humans.
LITERATURE CITED
1. Amlacher, E. 1961. Textbook of Fish Diseases. T.F.H. Publications, Neptune, N.J. 2. American Public Health Association. 1971. Standard methods for the examination of water and waste water, p. 660-662. 13 ed. American Public Health Association, Inc., New York. 3. Beijerinck, M. W. 1900. Schwefelwasserstoffbildung in den Stradtgraeben and Aufstellung der Gattung Aerobacter. Zentrabl. Bakteriol. Parasitenk. Infektionsker. Hyg. Abt. 2 6:193-206. 4. Breed, R. S., E. G. D. Murray, N. R. Smith (ed.). 1957. Bergey's manual of determinative bacteriology, 7th ed. Williams and Wilkins Co., Baltimore. 5. Bullock, G. L. 1964. Pseudomonadales as fish pathogens. Dev. Indust. Microbiol. 5:101-108. 6. Ewing, W. H., R. Hugh, and J. G. Johnson. 1961. Studies on the Aeromonas group p. 37. In Public Health Service Monogr. Communicable Disease Center, Atlanta, Ga.

Ferguson, W. W., and N. P. Henderson. 1947. Description of strain C27: a motile organism with the major antigen of Shigella sonnei phase I. J. Bacteriol. 54:179181. 8. Griffin, P. J., and S. F. Snieszko. 1951. A unique bacterium pathogenic for warm blooded and cold blooded animals. U.S. Fish Wildl. Ser. Fish. Bull. 68:187-190. 9. Kozlowski, F., and J. Antychowicz. 1966. Biochemical Properties of Aeromonas and Pseudomonas strains isolated from cases of carp dropsy. Bull. Vet. Inst. Pulawy 10:91-93. 10. Meeks, M. V. 1963. The genus Aeromonas: methods for identification. Amer. J. Med. Tech. 29:361-378. 11. Moller, V. 1955. Simplified tests for some amino acid decarboxylases and for the arginine dihydrolase system. Acta Pathol. Microbiol. Scand. 36:158-172. 12. Sanarelli, G. 1891. Ueber einen neuen Mikroorganismus
des Wassers, welcher fur Tiere mit veraenderlicher und konstanter Temperatur Pathogen ist. Zentrabl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. 2 9: 192-199; 222-228. Shotts, E. B., J. L. Gaines, L. Martin, and A. K. Prestwood. 1972. Aeromorns-induced deaths among fish and reptiles in an eutrophic inland lake. J. Amer. Vet. Med. Ass. 161:603-607. Taylor, W. I. 1965. Isolation of shigellae. I. Xylose lysine agars; new media for isolation of enteric pathogens. Amer. J. Clin. Pathol. 44:471-475. Taylor, W. I., and B. Harris. 1965. Isolation of shigellae. II. Comparison of plating media and enrichment broths. Amer. J. Clin. Pathol. 44:476-479. Von Gravenitz, A., and A. H. Mensch. 1968. The genus Aeromoonas in human bacteriology, a report of 30 cases and review of literature. N. Engl. J. Med. 278:245-249. Wohlgemuth, K., R. L. Pierce, and C. A. Kirkbride. 1972. Bovine abortion associated with Aeromonas hydrophila. J. Amer. Vet. Med. Ass. 160:1001. Zimmerman, 0. E. R. 1890. Die Bakterien unserer Trinkund Nutzwassers, part 1, p. 38-39. Chemnitz.

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