Introduction

An emerging problem in the pet fish industry is the increased antimicrobial resistance of common bacterial pathogens (Trust, 1972). Specifically, aeromonad resistance has become more obvious in the past several years. In our work, two aeromonad pathogens, Aeromonas hydrophila and Aeromonas sobria have been isolated from lesions or kidneys from domestic goldfish (Carassius auratus). Many antimicrobial agents have already been used to combat the problem of microbial infection, yet resistance appears to be on the rise with aeromonad strains isolated from imported tropical pet fish (Dixon, et al., 1990).

Abbott Laboratories (North Chicago, IL), has developed an aryl-fluoroquinolone antibacterial drug known as sarafloxacin hydrochloride, for use in aquaculture. Other types of antimicrobials such as aminoglycosides, nitrofurans, sulfa drugs and tetracycline are already being used in the ornamental fish industry.

The purpose of this study was to examine the in vitro antibiotic susceptibility patterns displayed by pathogenic bacteria.

Methods and Materials

Goldfish

Domestic goldfish, were obtained from a local wholesaler (Pan Ocean Aquarium, Inc. , Hayward, CA). Fish displaying either lesions or behavioral aberrations were first examined microscopically for external parasites on the gills or the fins.

Bacteria

The samples, obtained from either kidneys or external lesions were plated on Rimler-Shotts medium for 24 hours @30ºC (Shotts and Rimler, 1973). Oxidase positive yellow colonies were then sub cultured on Tryptic Soy Agar slants for further evaluation. The samples were tested on a motility medium, O-F glucose, glucose with a Durham tube and nutrient gelatin. In addition, novobiocin (5mcg) and 0-129 (0.1%) antimicrobial discs were also used as an assay to identify the bacteria to the genus level (Amos, 1985). For species identification, the Nonfermenter Test Strip kit (Analytab, Plainview, NY) was used. Antibiotic sensitivity was determined by plating the samples onto Mueller-Hinton agar (Remel, Sacramento, CA). After a 24 hour incubation 30ºC, the zones of inhibition was measured to determine resistance.

Susceptibility Discs

Results

Identification to the species level was obtained using the (Nonfermenter Test Strip) NFT system of the 60 isolates, 40 were identified as A. hydrophila, 16 as A. sobria and 4 as A. spp. All of the colonies were resistant to both novobiocin and 0/129.

With respect to antimicrobial susceptibility, four of the twelve antibiotics tested showed antimicrobial resistance, including ampicillin, tetracycline, erythromycin, and the triple sulfa drug. The aryl-fluoroquinolone, sarafloxacin hydrochloride, has proved its efficacy as an antimicrobial drug, with only 1 resistant strain isolated.

Discussion

Despite the increasing frequency in resistance to antibiotics exhibited by tropical fish, it appears that many of the antibacterial are effective against domestic strains of bacteria such as A.hydrophila and A.sobria. The fear, if this indeed is the case regarding foreign and domestic strains of the same organism, is that plasmid mediated transfer of resistant genes would significantly decrease the frequency of antibiotic susceptibility. The resistance to quinolones is carried on the chromosomal DNA and would subsequently take longer to be transferred. Unfortunately, antibiotics such as ampicillin and tetracycline have apparently run their course as effective treatments to microbial diseases (Van Impe, 1977). Yet, there is hope that the development of new antimicrobials, such as the aryl-fluoroquinolone sarafloxacin hydrochloride, will prove to be efficacious antibacterial treatments.

Acknowledgements

Funding for this study was provided by Abbott Laboratories North Chicago, IL and the Western World Pet Supply Association, South Pasadena, CA.

References

1.  Amos, L.(ed.) 1985. Procedures for the detection and identification of certain fish pathogens, 3rd Edition. Fish Health Section, American Fisheries Society, Corvalis, OR.

2.  Dixon, B., Evelyn, F. and Yamashita, J., 1990. Antibiotic Resistance of Aeromonas spp. isolated from Tropical Fish Imported from Singapore. J. Aquatic Health Vol. 2, #4 pp. 295-297.

3.  Trust, T.J., 1972 Inadequacy of Aquarium Antibacterial Formulations for the inhibition
of potential pathogens of fish. Can J. Fis. Res. Board 29 #10.

4.  Shotts, E.B., Rimler 1973 Medium for The Isolation of Aeromonas hydrophila. Appl. Micro. 26:550-553.

5.  Van Impe, J., 1977 Etude de 50 souches d'Aeromonas hydrophila isolées du poissons. Ann. Biol. Clin.,35,329-337.