Development Of Oxytetracycline Resistance In Rainbow Trout (Oncorhynchus mykiss) Gut Flora And Biofilter Flora In A Recirculating System                    W. Bane Schill1, Graham Bullock2, and Erik Burchard2   1USGS, National Fish Health Research Laboratory, 1700 Leetown Road, Kearneysville, WV, 25430; 2Conservation Fund, Freshwater Institute, P.O. Box 1889, Shepherdstown, WV, 25443     The increasing numbers of antibiotic resistant pathogenic bacteria caused by increased use of antibiotics has caused concern in the scientific community.  The use of antibiotics in aquaculture has been suggested as contributing to the problem. We studied the effect of feeding a 14-day regimen of oxytetracycline (OTC) to rainbow trout cultured in a recirculating system on development of resistance in gut flora and biofilter flora. Total bacterial counts were carried out on 12 fecal pools (5 trout/pool) and 6 biofilters before feeding OTC medicated diet (3 grams/ 100 lbs. Trout/day).  Counts were repeated on days 3, 7, and 14 of OTC feeding; and on days 3, 7, 14, 21, 28, and 42 days post OTC feeding. Samples were cultured on R2A and Mueller Hinton (MH) media with and without 100 μg/ml OTC, and enumerated by the drop plate procedure after 72 hr incubation at 23ºC. Fecal bacterial counts were equivalent with either media, but R2A consistently yielded higher recovery for biofilter organisms.  Before feeding, fecal pools yielded 0.11% resistant colonies (expressed as average % of total heterotrophic count on the two media). By day 7 of OTC feeding resistance was 61.25%, and increased to 73.65% by day 14. After completion of OTC feeding, resistance rapidly dropped to 35.9% by day 3 and 1.26% by day 21. Unlike the large increase in resistance found in fecal bacteria after 14 days of OTC feeding, resistant bacteria in biofilters only increased to a maximum of 1.79% on R2A.  By day 21 post OTC feeding, these levels of resistance dropped to 0.17%. Development of multiple antibiotic resistance (MAR) in fecal bacteria was observed after 14 days of OTC feeding.  Before OTC feeding, 22 of 48 OTC resistant fecal isolates from R2A and 11 of 41 resistant isolates from MH were also resistant to chloramphenicol, kanamycin, streptomycin, and ampicillin at 50 μg/ml.  MAR was confined primarily to two pools, indicating that MAR organisms were probably donated to the pools by single fish.  After 14 days of OTC feeding, however, 192 of 192 isolates from both media and all pools were MAR positive.  A second cycle of antibiotic feeding produced a very much diminished (as compared to the first cycle) increase in resistant bacteria.  Possible mechanisms for this result are explored and preliminary results of another study following the gut flora of individual fish are discussed.