Introduction

It is well established in the pet fish industry that bacterial infections are responsible for heavy losses from the farm level to the hobbyist tank. Over the years, many bacterial pathogens have been isolated and identified, of these one of the most important is Aeromonas hydrophila. Whether as a primary infection in wounds or a secondary problem following the stresses of temperature change, handling or poor water quality, Aeromonas has emerged as the most common bacterial pathogen. This pathogen is common not only in freshwater tropicals, but also in baitfish and foodfish such as Channel Catfish. In an intensive study some years ago, 73 bags of fishes from southeast Asia were examined for the presence of bacteria as well as other parasites (Gratzek et. al, 1978). Of these 15% of fish had aeromonad bacteremia and 14% of the shipping water contained this organism, clearly establishing this bacterium as a major disease problem in need of control (Shotts et a-I., 1976). By this time, the industry had already adopted the use of many antimicrobial agents ranging from aminoglycosides (Neomycin, Kanamycin) to tetracyclines, sulfa drugs, nitrofurans and others. However, the study by Gratzek et a]. (1978), did not include individual antibiotic susceptibility patterns (antibiograms) to determine which drugs were most useful.

Over the years, tropical fish wholesalers and retailers have felt there is increasing bacterial resistance to standard antibiotic treatments, particularly in fish shipped from Asia. Some of these implications have been confirmed by scientific study. Several recent studies from Japan have substantiated that chloramphenicol and tetracycline resistant Aeromonas hyd was the principal bacterium isolated from the intestinal tracts of Ayu. Further drug resistant bacteria isolated from ponds corresponded to those isolated from the intestinal tracts (Aoki et al. 1980). Other Japanese workers have stated that all of the Aeromonas hydrophila isolated from Carp, Ayu, Eel, Rainbow Trout and Yamame carried resitance (Hayashi 1985). In this country, reports from an eel aquaculture facility in South Carolina, define a positive correlation between the use of nitrofurazone and the isolation of nitrofurazone resistant strains of Aeromonds h .hydrophia (Davis and Hayasaka 1983). There are other reports of bacteria drug resistance in foodfish including Atlantic Salmon in Scotland and Carp in Germany (Hastings and McKay 1987). However, most of this resistance in evidenced in foodfish culture with surprisingly little data on imported tropicals. In efforts to curtail fish losses, individuals in the petfish industry are left to try various combinations of antibiotics or unknowingly try ineffective antibiotics. In addition to severely stressing fish, combinations of drugs may actually exacerbate the problem by selecting for resistant bacteria.

This study was undertaken to determine the magnitude and scope of the bacterial resistance problem, in the hope of providing wholesalers with information to treat imported tropical fish more effectively.

Materials and Methods

To determine the scope of the bacterial resistance problem, over 100 tropical fish imported from Singapore were sampled. Following isolation and identification of Aeromonas, eleven commonly used antimicrobials and one new fluoroquinolone were evaluated for in vitro efficacy.

Materials and Methods

Fish: Tropical freshwater fish imported from Singapore were obtained from a local wholesaler (Pan Ocean Aquarium Inc., Hayward, CA) Fish displaying gross external lesions were sampled directly from the lesion and kidney. Fish without lesions, but displaying behavioral changes were sampled from the kidney. All fish were examined for the presence of external and internal parasites. All classes of fish were utilized in the study, including Anabantids, Characins, Cichlids, Catfish and others.

Bacteria: The samples were plated on R-S medium and incubated at 35°C, for 24 hours (Shotts & Rimmler 1973). Following isolation, oxidase positive yellow colonies presumptive identified as Aeromonas spp. were inoculated into API 20E and NFT (Analytab Products, Plainview, NY.) systems for species identification. Identified samples were streaked onto Mueller-Hinton agar (Remel, Sacramento CA), for antibiotic susceptibility testing. This procedure utilized the standard Kirby-Bauer disc diffusion technique. Following incubation at 35°C for 24 hours, inhibition zone size was measured and classified according to resistant or sensitive (Amos 1985).

The following susceptibility discs were used: Erythromycin (E) 0.015 mg, Nalidixic Acid (NA) 0.03 mg, Neomycin (N) 0.03 mg, Sulfamethoxazole 25 mcg/Trimethoprim 25 mcg (SXT) obtained from General Diagnostics Morris Plains, NJ, 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, MI. Oxolini Acid (OX) 2 mcg and Ormetoprim 1.2 mcg/ Sulfadimethoxine 23.8 mcg (Romet) Supplied by BBL Cockneysville, MD, Sarafloxacin (SF) 2 mcg was supplied by Abbott Laboratories, North Chicago, IL.

Results

Isolate identification by the API 20E system was less than adequate. Many isolates did not key to an API code number, however the NFT system did key these organisms to the species level. Some isolates that keyed to Aeromonas hydrophila on the API 20E system subsequently keyed to other species on the NFT system. The NFT determinations were used as the more valid identification method. Of the seventy isolates identified to genus as Aeromonas, twenty were speciated as A. caviae, twenty as A. sobria and thirty as A. hydrophila by the NFT method. A. hydrophila with A. caviae or A. sobria were isolated in concomitant infections in some samples. A. sobria and A. caviae were not isolated as concomitant infections. All three species were also individually isolated from infections.

As evidenced by the data, over half of the seventy isolates were resistant to seven of the twelve antibacterials tested. A. sobria was the most resistant, with over half of the twenty isolates showing resistance to nine of the twelve drugs. As a group, the quinoline antibacterials were the most effective. The new fluoroquinolone, sarafloxacin, showed the lowest level of bacterial resistance. Of the seventy isolates, only six showed resistance by disc testing to sarafloxacin. Determination of minimum inhibitory concentrations of sarafloxacin for these isolates showed them to be of borderline susceptibility. Neomycin also still proved to be efficacious in vitro, with only eighteen of seventy isolates showing resistance.

Discussion

In view of these data, it is clear that resistance to commonly used antibiotics is emerging rapidly among Aeromonas population infecting Asian imported tropical fish. Over half of the seventy Aeromonas isolates obtained from infections, were resistant to seven of the twelve antibacterial drugs tested.

Acknowledgements

This research was funded by grants from Abbott Laboratories, North Chicago, IL, and the Western World Pet Suppliers Association, South Pasadena, CA.

References

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