THE JOURNAL OF THE
WORLD AQUACULTURE SOCIETY
Vol. 24, No. I March, 1993

Antibacterial Drug Resistance in Aeromonas spp.
Isolated from Domestic Goldfish and
Koi from California
BEVERLY A. DIXON' AND GERARD ISSVORAN
Department of Biological Sciences, California State University, Hayward, California 94542 USA

Prior in vitro studies in the laboratory have shown that Aeromonas spp. isolated from imported Asian tropical fish develop resistance to commonly used antibiotics (Dixon et al. 1990). Of the 70 isolates obtained from lesion and kidney samples, over half were resistant to 7 of the 12 antibiotics tested in that study. In some cases the bacteria were resistant to 9 of the 12 antibiotics tested. Only six (8.5%) isolates showed resistance to sarafloxacin hydrochloride, a new antibacterial compound developed by Abbott Laboratories (North Chicago, Illinois).
     A comparable study was performed on domestic goldfish Carassius auratits including common, fancy and koi Carassius auratus x Cyprinus carpio varieties. Goldfish make up a large part of both the baitfish and ornamental fish markets in this country. Fancy varieties of goldfish and koi are commonly imported into the U.S. from China, Japan and other Asian countries, nevertheless a large domestic industry also exists.
     Goldfish and koi are susceptible to infection with Aeromonas hydrophila, especially during the warmer water temperatures of summer, when secondary infections result from high parasite loads. In addition, goldfish are susceptible to infection with Aeromonas salmonicida referred to as atypical salmonicida or Goldish Ulcer Disease (GUD). GUD can cause massive die-offs in the early spring when water temperatures begin to rise (Elliott and Shotts 1980; Shotts et al. 1980).
     The present study was undertaken to determine the antibiotic susceptibility of the motile Aeromonas spp. isolated from common and fancy varieties of goldfish and koi, and to provide useful information on the efficacy of present disease treatments in the domestic goldfish producing industry.

Materials and Methods
  Domestic goldfish raised in the San Joaquin Valley of California were used in this study. Following isolation and identification of Aeromonas spp. from these fish, 11 commonly used antimicrobial compounds and one new aryl- fluoroquinolone were evaluated for in vitro efficacy.
     Over the course of nine months, test fish were obtained from a local wholesaler (Pan Ocean Aquarium Inc., Hayward, California). Lesions and/or kidney of fish displaying gross external lesions were sampled for the presence of bacteria. The bacterial samples were plated on Rimler-Shotts medium (Shotts and Rimler 1973) and incubated at 30 C for 24 h. Isolated colonies were identified according to the American Fisheries Society Fish Health Section Bluebook Guidelines (Amos 1985). Oxidase positive isolates were tested for motility, sensitivity to novoblocin (5 mcg) and 0/ 129 (0. 1 %), growth in salt free nutrient gelatin, and fermentation of glucose on OF medium. Colonies presumptively identified as Aeromonas were inoculated into the Rapid Nonfermenter Test strip (NFT) system (Analytab Products, Plainview, New York) for species identification. Identified isolates were plated onto Mueller-Hinton agar (Remel, Sacramento, California), for antibiotic susceptibility testing, using the Kirby-Bauer disk-agar diffusion method (Lennette 19 8 5).

TABLE 1. Antibiotic resistance among 60 isolates of Aeromonas spp. from domestic goldfish

Antibiotic                                                                                     No. (%) of resistant isolates

     Susceptibility to antibiotics and the new arylfluoroquinolone compound was determined. Following incubation at 30 C for 24 hours, inhibition zone sizes were measured. The following susceptibility discs were used: erythromycin (E) 0.015 mg, nalidixic acid (NA) 0.03 mg, neomycin (N) 0.03 mg, and sulfamethoxazole 25 mcg/trimethoprim 25 mcg (SXT) obtained from General Diagnostics (Morris Plains, New Jersey), and nitrofuradantoin (FD) 0.3 mg, triple sulfa (SSS) 0.3 mg, tetracycline (TE) 0.03 mg, trimethoprim (TMP) 5 mcg, and ampicillin (AM) 10 mcg obtained from Difco Laboratories (Detroit, Michigan). Oxolinic acid (OX) 2 mcg and ormethoprim 1.2 mcg/sulfadimethoxine 23.8 mcg (Romet-30®) were supplied by Baltimore Biological Laboratories (Cockleysville, Maryland), and sarafloxacin HCl (SF) 2 mcg was supplied by Abbott Laboratories (North Chicago, Illinois).

Results

     Sixty bacterial isolates were identified from lesion and kidney samples obtained from domestic goldfish and koi. Of these, 41 were identified by the NFT method as A. sobria, 15 as A. hydrophila, and 4 were identified only to the genus level.
     The antibiogram results obtained from these motile aeromonad isolates are shown in Table 1. Except for ampicillin, all the other antibacterial compounds tested still showed activity against the bacteria. Of these drugs, tetracycline was the only one to which more than half (60%) of the bacterial population was resistant. These data indicate that the effectiveness of tetracycline is questionable for treating aeromonad infections in this population of fish. Only one bacterial isolate, identified as A. sobria, was resistant to the new fluoroquinolone sarafloxacin HCl.

Discussion

     Whcthcr for food, bait or beauty, the selective breeding of "carp" has become profitable on a wide scale. For the ornamental trade in particular, common and fancy varieties of goldfish and koi are bred throughout the world. Antibiotic resistance in A. salmonicida, responsible for goldfish ulcer disease, has already been demonstrated in salmonid culture (Wood et al. 1986). Resistance to oxolinic acid and tetracycline has been reported in salmonid furunculosis infections in Scotland, England, Germany, Norway and Chile (Hastings and McKay 1987; Tsoumas et al. 1989). Japanese workers have described A. hydrophila isolates from carp culture that display antibiotic resistancc (Aoki et al. 1980; Hayashi 1985). Aeromonad resistance to tetracycline was demonstrated in this study in 60% of the isolates. Although oxolinic acid is not widely used in the United States, 7% of the isolates in this study were resistant. Compared with a previous study (Dixon et al. 1990), domestic aeromonad isolates have not yet developed the level of antimicrobial resistance that was demonstrated in isolates from petfish imported from the Far East. In that study 96% of the Aeromonas spp. isolated from petfish imported from Singapore were resistant to tetracycline. Of interest to the foodfish industry, 67% of these isolates were resistant to Romet-30®, whereas only 12% of the domestic isolates were resistant. Tetracycline and Romet-30® are the only two antibacterial compounds approved for use in the U.S. for foodfish culture. Multiple resistance was common among the imported isolates, over half of which showed resistance to 7 of the 12 antimicrobial compounds tested (Dixon et al. 1990).
     In view of the data obtained in this study, it appears that although antibiotic resistance is not yet prevalent in this population of domestic aeromonad isolates, the potential exists for a rapid emergence of resistance. Of particular interest is the problem of possible transfer of resistance between bacterial strains from ornamental and foodfish culture. Tetracycline resistance has already been observed in aeromonad isolates from cultured channel catfish (Ictalurus punctatus) and their environments in Alabama and Mississippi (DePaola et al. 19 8 8). Furthermore, Aeromonas spp. are being increasingly isolated from both healthy and immunocompromised human patients (Janda and Duffey 1988). Multiple resistance and differences in resistance patterns were observed between strains of isolates from humans from different geographical locations (Chang and Bolton 1987; Motyl et al. 1985). In light of the bacterial resistance problems already recognized in human and veterinary medicine, this problem needs to be studied further among bacterial pathogens occurring in the ornamental fish industry.

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