Introduction

Studies in our laboratory have shown that bacterial resistance to commonly used antibiotics is an emerging problem in the ornamental fish industry. In a recently completed study, isolates of Aeromonas spp. from tropical fish imported from Singapore showed multiple resistances to tetracycline, sulfa drugs, erythromycin, and occasionally to quinolone antibacterials (Dixon et al. 1990). In addition to resistance in ornamental fish populations, antimicrobial resistance was also documented in bacteria isolated from food fish (Bullock 1984; MacMillan 1985; Ganzhorn 1987; Hastings and McKay 1987; Taylor 1987; Stamm 1989). These reports document aeromonad resistance to Terramycin and Romet®, the only two antibiotics approved for use on foodfish in this country.

Due to the increasing number of reports, many of the commonly used antimicrobials may no longer prove efficacious for the treatment of this gram negative fish pathogen. It may be necessary at this time to begin screening antimicrobial compounds for efficacy against fish pathogens. This study was undertaken to determine the in vitro efficacy of Naxcel® for clinical isolates of Aeromonas spp. isolated from ornamental fish.

Naxcel®, a sodium salt of ceftiofur, is a broad spectrum B-lactamase resistant cephalosporin. As a third generation cephalosporin, Naxcel® provides bactericidal activity resulting from inhibition of cell wall synthesis. Naxcel® is currently marketed by The Upjohn Co. for treatment of bovine respiratory disease (pneumonia, shipping fever) associated with Pasteurella hemolytica and P. multocida infections. Ceftiofur has also demonstrated excellent in vitro activity against gram negative bacterial pathogens such as Escherichia coli, Salmonella typhimurium and Haemophilus pleurogneumonia (Yancy et al 1987).

This study was undertaken to determine the in vitro effectiveness of Naxcel® for Aeromonas spp. isolated from ornamental fish.

Materials and Methods

Lesion and/or kidney samples from ornamental fish were plated on Rimle-Shotts medium for primary isolation of Aeromonas spp., and incubated at 30C. Isolated colonies were identified according to Fish Health Section Bluebook guidelines (Amos 1985). Oxidase positive isolates were tested for motility, sensitivity to novobiocin (5mcg) and 0/129 (0.1%), growth in salt free nutrient gelatin and fermentation of glucose on OF medium. Colonies identifying to the genus were inoculated into the Nonfermenter Test strip (NFT) system (Anatylab Products, Plainview, NY) for species identification. Once identified, colony isolates were plated onto Mueller-Hinton agar (Remel, Sacramento, CA) for determination of in vitro antibiotic sensitivity. The standard Kirby-Bauer disk diffusion method was used to determine antibiotic sensitivity to commonly used antibiotics and Naxcel R. Following incubation at 30C for 24 hours, inhibition zone sizes were measured. The following susceptibility disks were used: erythromycin 0.015mg, nalidixic acid 0.03mg, neomycin 0.03mg, sulfamethoxazole 25mcg/trimethoprim 25mcg obtained from General Diagnostics (Morris Plains, NJ), nitrofurantoin 0.3mg, triple sulfa 0.3mg, tetracycline 0.03mg, trimethoprim 5mcg and ampicillin 10mcg obtained from Difco Laboratories (Detroit, MI). Oxolinic acid 2mcg and ormetoprim 1.2mcg/ sulfadimethoxine 23.8mcg (Romet R) supplied by Baltimore Biological Laboratories (Cockneysville, MD), and Naxcel R 3Omcg supplied by Upjohn (Kalamazoo, MI). Minimum inhibitory concentrations (MIC) were determined by the Veterinary Diagnostic Laboratory System, University of California, Davis, CA.

Results

Of the 31 isolates identified, 19 were identified as A. sobria, 11 as A. hydrophila and 1 as A. sp., by the Nonfermenter Test strip system. None of the isolates was resistant to Naxcel® as determined by zone of inhibition size measurements. MIC determinations ranged from <= 0.25µg/ml to 1.0µg/ml, well within the susceptible range. However, all of the isolates were resistant to ampicillin, 71% were resistant to tetracycline. Resistance to all the other antimicrobials was less than 29%.

Conclusions

Naxcel R appears to be highly effective in vitro against Aeromonas spp. isolated from ornamental fish. Further in vivo study is needed to determine the potential for Naxcel® as an efficacious treatment for aeromonad infections of fish.

Acknowledgements

Funding for this study was provided by The Upjohn Company, Kalamazoo, MI.

References

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2.  Bullock, G. 1984. Enteric red mouth disease of salmonids. U.S. Fish and Wildlife Service Fish Disease Leaflet 67.

3.  Dixon, B.A., J. Yamashita and F. Evelyn. 1990. Antibiotic resistance of Aeromonas spp. isolated from tropical fish imported from Singapore. J. Aq. Animal Health 2:4 295-297.

4.  Ganzhorn, J. 1987. Aeromonas salmonicida, apparent drug resistance to ormetoprim-potentiated sulfadimethoxine. American Fisheries Society, Fish Health Section Newsletter 15:4 (Bethesda, MD).

5.  Hastings, T., and A. McKay. 1987. Resistance of Aeromonas salmonicida to oxolinic acid. Aquaculture 61:165-171.

6.  MacMillan, J. 1985. Infectious Diseases. Pages 405-496 i C.S. Tucker, editor. Channel Catfish Culture. Elsevier, Amsterdam.

7.  Stamm, J.M. 1989. In vitro resistance by fish pathogens to aquacultural antibacterials, including the quinolones Difloxacin (A-56619) and Sarafloxacin (A-566120). J. Aq. Animal Health 1:135-141.

8.  Taylor, P. 1987. Antibiotic resistance to Romet®-30 in bacterial infections of catfish. American Fisheries Society, Fish Health Section Newsletter 15:4 (Bethesda, MD).

9.  Yancy, R.J. et al. 1987. Ceftiofur sodium, a broad-spectrum cephalosporin: evaluation in vitro and in vivo in mice. Am. J. Vet. Res. 48:7 1050-1053.