J. appl. Bact. 1975,38, 67-69
An Improvement in the Technique for the Presumptive Identification of Aeromonas salmonicida J. M. -r. HAMILTON-MILLER AND J. OGUNNARIWO Department of Medical Microbiology, Royal Free Hospital, Gray's Inn Road, London, W.C.I , England Received 29 May 1974 and accepted I6 September 1974
AEROMONAS SALMONICIDA is the aetiological agent of furunculosis and, as such, is an important fish pathogen (Scott, 1968). Two simple presumptive tests for A . salmonicida have been described. The first, a rapid method, is due to Griffin, Snieszko & Friddle (1953) and depends upon the production of a deep purple-black colour when a chromogenic reagent is added to an overnight culture of the organism. Close examination of this paper,,however, reveals the misconception that this purple-black colour is qualitatively the same as the brownish-red pigment known to be produced spontaneously by A . sai'monicida after 2-3 days at 22". As the chromogenic reagent is pphenylenediamine, the purple colour is merely the result of a positive oxidase test. At the time of this work the oxidase test, although having been described many years previously (Gordon & McLeod, 1928), had not come into general diagnostic use; it was the modification of Kovacs (1956) that eventually brought this about. Thus, the rapid test described by GrifEn et al. (1953), far from being presumptive for A . salmonicida, will fail to differentiate, among Gram negative bacilli, between the following oxidase-positive genera : Actinobacillus, Aeromonas, Alcaligenes, Brucella, Flavobacterium, Moraxella, Pasteurella, Pseudomonas and Vibrio (Cowan & Steel, 1965). Even motility testing, suggested as a useful secondary procedure by Griffin et al. (1953), will eliminate only the motile genera Alcaligenes, Pseudomonas and Vibrio from this list. The second presumptive test for A. salmonicida depends upon the production of brown pigment within 2-3 days on furunculosis agar. We have investigated recently 6 strains of Pseudomonas ueruginosu which produce melanin (Ogunnariwo & HamiltonMiller, in press) arid our findings with these organisms suggest that such strains could be confused readily with A. salmonicida on furunculosis agar. It is the purpose of the present note to describe the source of such possible confusion and to suggest a way in which it may be avoided.
Materials and Methods Organisms Pseudomonas aeruginosa RFH 355,902 and 908 were isolated from clinical specimens sent to the routine laboratory of this hospital. Pseudomonas aeruginosa NCTC 5083, 6749 and 10561 arid A . salmonicida NCTC 10402 were obtained from the National 1671
68
J. M. T. HAMILTON-MILLER AND J. OGUNNARIWO
Collection of Type Cultures. All 6 strains of Ps. aeruginosa produce pyomelanin and the pigment from A. salmonicida also behaves like melanin.
Culture medium Furunculosis agar (Difco) was used. It has the following composition (g/l) : tryptone, 10; yeast extract, 5 ; L-tyrosine, 1; NaC1,2.5; agar, 15; distilled water, 1000 ml.
Motility testing The organisms were inoculated into peptone water, incubated for 4 h at 37" and examined with a phase-contrast microscope.
Results and Discussion Rapid test with p-phenylenediamine Strains were inoculated on slopes of the medium described by Griffin et al. (1953) and incubated overnight (18 h) at room temperature. The test was positive with A. salmonicida. It was not applicable, however, to the strains of Ps. aeruginosa, as spontaneous pigmentation was already so marked that any purple colour would be masked. All 6 strains of Ps. aeruginosa were, however, oxidase positive on media, such as blood agar and MacConkey agar, which are not conducive to pigment production.
Pigmentation on furunculosis agar as a function of temperature Aeromonas salmonicida showed little growth and no pigmentation after 24 h incubation at room temperature; pigmentation was evident after 48 h. All 6 strains of Ps. aeruginosa had started to produce pigment after 18 h incubation at room temperature and the medium became very dark after 24 h. At 37" A . salmonicida grew very poorly even after prolonged incubation and did not produce any pigment; on the other hand, the Ps. aeruginosa strains grew luxuriantly overnight and produced more pigment than at room temperature. Thus, differentiation of A. salmonicida from melanogenic strains of Ps. aeruginosa cannot be made at room temperature using furunculosis agar as suggested (Anon, 1968). We suggest that 2 slopes are inoculated, one incubated at room temperature and the other at 37"; if both slopes turn brown, the organism is not A. salmonicida. Motility
AU our 6 strains of Ps. aeruginosa were motile. This could be used to differentiate them from A . salmonicida as a second-stage test following the demonstration of pigment production. It should be noted, however, that a small but significant proportion of melanin-producing Ps. aeruginosa strains are non-motile: Elston (1968) and Yabuuchi & Ohyama (1972) reported incidences of 25 % and 10%, respectivefy, of non-motile strains among melanin producers.
IDENTIFICATION OF AEROMONAS SALhlONICIDA
69
References ANON.(1968). Difco Supplementary Literature. Furunculosis agar, p. 159. Michigan, U.S.A.: Difco Laboratories. COWAN,S. T. & STEEL,K. J. (1965). Manual for the IdentiJication of Medical Bacteria. Cambridge: University Press. ELSTON, H. R. (1968). Melanogenic strains of Pseudomonas aeruginosa in biological specimens. Am. J. med. Technol. 34, 189. GORDON, J. & MCLEOD,J. W. (1928). The practical application of the direct oxidase reaction in bacteriology. J. Path. Bact. 31, 185. GRIFFIN, P. J., SNIESZKO, S. F. & FRIDDLE, S. B. (1953). A new adjuvant in the diagnosis of fish furunculosis caused by Bacterium salmonicida. Vet. Med. 48, 280. KOVACS, N. (1956). Identification of Pseudomonaspyocyanea by the oxidase reaction. Nature, Lond. 178, 703. SCOTT,M. (1968). The pathogenicity of Aeromonas salmonicida (Griffin) in sea and brackish waters. J. gen. Microbial. 50, 321. E. & C~HYAMA,A. (1972). Characterization of ‘pyome1anin’-producing strains YABUUCHI, of Pseudomonas aeruginosa. Int. J. syst. Bact. 22, 53.
An Improvement in the Technique for the Presumptive Identification of Aeromonas salmonicida J. M. -r. HAMILTON-MILLER AND J. OGUNNARIWO Department of Medical Microbiology, Royal Free Hospital, Gray's Inn Road, London, W.C.I , England Received 29 May 1974 and accepted I6 September 1974
AEROMONAS SALMONICIDA is the aetiological agent of furunculosis and, as such, is an important fish pathogen (Scott, 1968). Two simple presumptive tests for A . salmonicida have been described. The first, a rapid method, is due to Griffin, Snieszko & Friddle (1953) and depends upon the production of a deep purple-black colour when a chromogenic reagent is added to an overnight culture of the organism. Close examination of this paper,,however, reveals the misconception that this purple-black colour is qualitatively the same as the brownish-red pigment known to be produced spontaneously by A . sai'monicida after 2-3 days at 22". As the chromogenic reagent is pphenylenediamine, the purple colour is merely the result of a positive oxidase test. At the time of this work the oxidase test, although having been described many years previously (Gordon & McLeod, 1928), had not come into general diagnostic use; it was the modification of Kovacs (1956) that eventually brought this about. Thus, the rapid test described by GrifEn et al. (1953), far from being presumptive for A . salmonicida, will fail to differentiate, among Gram negative bacilli, between the following oxidase-positive genera : Actinobacillus, Aeromonas, Alcaligenes, Brucella, Flavobacterium, Moraxella, Pasteurella, Pseudomonas and Vibrio (Cowan & Steel, 1965). Even motility testing, suggested as a useful secondary procedure by Griffin et al. (1953), will eliminate only the motile genera Alcaligenes, Pseudomonas and Vibrio from this list. The second presumptive test for A. salmonicida depends upon the production of brown pigment within 2-3 days on furunculosis agar. We have investigated recently 6 strains of Pseudomonas ueruginosu which produce melanin (Ogunnariwo & HamiltonMiller, in press) arid our findings with these organisms suggest that such strains could be confused readily with A. salmonicida on furunculosis agar. It is the purpose of the present note to describe the source of such possible confusion and to suggest a way in which it may be avoided.
Materials and Methods Organisms Pseudomonas aeruginosa RFH 355,902 and 908 were isolated from clinical specimens sent to the routine laboratory of this hospital. Pseudomonas aeruginosa NCTC 5083, 6749 and 10561 arid A . salmonicida NCTC 10402 were obtained from the National 1671
68
J. M. T. HAMILTON-MILLER AND J. OGUNNARIWO
Collection of Type Cultures. All 6 strains of Ps. aeruginosa produce pyomelanin and the pigment from A. salmonicida also behaves like melanin.
Culture medium Furunculosis agar (Difco) was used. It has the following composition (g/l) : tryptone, 10; yeast extract, 5 ; L-tyrosine, 1; NaC1,2.5; agar, 15; distilled water, 1000 ml.
Motility testing The organisms were inoculated into peptone water, incubated for 4 h at 37" and examined with a phase-contrast microscope.
Results and Discussion Rapid test with p-phenylenediamine Strains were inoculated on slopes of the medium described by Griffin et al. (1953) and incubated overnight (18 h) at room temperature. The test was positive with A. salmonicida. It was not applicable, however, to the strains of Ps. aeruginosa, as spontaneous pigmentation was already so marked that any purple colour would be masked. All 6 strains of Ps. aeruginosa were, however, oxidase positive on media, such as blood agar and MacConkey agar, which are not conducive to pigment production.
Pigmentation on furunculosis agar as a function of temperature Aeromonas salmonicida showed little growth and no pigmentation after 24 h incubation at room temperature; pigmentation was evident after 48 h. All 6 strains of Ps. aeruginosa had started to produce pigment after 18 h incubation at room temperature and the medium became very dark after 24 h. At 37" A . salmonicida grew very poorly even after prolonged incubation and did not produce any pigment; on the other hand, the Ps. aeruginosa strains grew luxuriantly overnight and produced more pigment than at room temperature. Thus, differentiation of A. salmonicida from melanogenic strains of Ps. aeruginosa cannot be made at room temperature using furunculosis agar as suggested (Anon, 1968). We suggest that 2 slopes are inoculated, one incubated at room temperature and the other at 37"; if both slopes turn brown, the organism is not A. salmonicida. Motility
AU our 6 strains of Ps. aeruginosa were motile. This could be used to differentiate them from A . salmonicida as a second-stage test following the demonstration of pigment production. It should be noted, however, that a small but significant proportion of melanin-producing Ps. aeruginosa strains are non-motile: Elston (1968) and Yabuuchi & Ohyama (1972) reported incidences of 25 % and 10%, respectivefy, of non-motile strains among melanin producers.
IDENTIFICATION OF AEROMONAS SALhlONICIDA
69
References ANON.(1968). Difco Supplementary Literature. Furunculosis agar, p. 159. Michigan, U.S.A.: Difco Laboratories. COWAN,S. T. & STEEL,K. J. (1965). Manual for the IdentiJication of Medical Bacteria. Cambridge: University Press. ELSTON, H. R. (1968). Melanogenic strains of Pseudomonas aeruginosa in biological specimens. Am. J. med. Technol. 34, 189. GORDON, J. & MCLEOD,J. W. (1928). The practical application of the direct oxidase reaction in bacteriology. J. Path. Bact. 31, 185. GRIFFIN, P. J., SNIESZKO, S. F. & FRIDDLE, S. B. (1953). A new adjuvant in the diagnosis of fish furunculosis caused by Bacterium salmonicida. Vet. Med. 48, 280. KOVACS, N. (1956). Identification of Pseudomonaspyocyanea by the oxidase reaction. Nature, Lond. 178, 703. SCOTT,M. (1968). The pathogenicity of Aeromonas salmonicida (Griffin) in sea and brackish waters. J. gen. Microbial. 50, 321. E. & C~HYAMA,A. (1972). Characterization of ‘pyome1anin’-producing strains YABUUCHI, of Pseudomonas aeruginosa. Int. J. syst. Bact. 22, 53.
