Document Type

Master's Research

Degree Name

Master of Science

Department

Biology

Advisor(s)

Shree S. Dhawale, Ahmed Mustafa

Date of Award

5-2013

Abstract

Antibiotics and antibiotic resistant bacteria have been detected in the aquatic system around the world. We wanted to know whether the wild fish population from Cedar Creek watershed in Indiana and reference site in Michigan had any antibiotic resistant microbes in their gut samples. Additionally we wanted to test hatchery-raised fish for the same reason. The main objectives were to 1) isolate antibiotic resistant bacteria that might be present in the fish guts, 2) determine the antibiotic resistance phenotype of resistant isolates, and 3) to test for the presence of plasmids in selected resistant isolates. Freshwater field fish species were creek chubs (Semotilus atromaculatus) and mottled sculpins (Cottus bairdi), and hatchery-raised fish were tilapia (Oreochromis niloticus) and coho salmon (Oncorhynchus kisutch). Creek chubs and mottled sculpins were collected from 1) two tributaries of Cedar Creek- Matson Ditch and Swartz Ditch in Indiana that drain row-crop agricultural lands, and 2) a reference site in the East Branch of the St. Joseph River near Michigan that drains primarily forested land of the Lost Nation Game Reserve. Coho salmon and tilapia were obtained from hatcheries and maintained in a fish laboratory at IPFW.

The antibiotic resistant bacteria from the fish gut were isolated on the basis of their resistance to ampicillin. Out of 70 fish included in this study 50 fish yielded antibiotic resistant isolates. From each fish except one, 5 bacterial colonies were isolated giving a total of 248 isolates. The results indicated prevalence of antibiotic resistant bacteria in the gut samples both in the field fish and in the hatchery-raised fish. The Matson Ditch fish had the highest percentage (94%) of ampicillin resistant bacteria among field sites that were sampled. Among two field fish species, creek chubs had more antibiotic resistant bacteria (85%) than mottled sculpin (67%). In case of hatchery-raised fish, 75% tilapia and 38% coho salmon had antibiotic resistant bacteria.

All 248 bacterial isolates were further analyzed to determine their antibiotic resistance pattern by the Kirby Bauer method using six additional antibiotics -penicillin (pen), vancomycin(van), chloramphenicol (cap), tetracycline (tet), streptomycin (str) and gentamicin (gen). Since all isolates were Gram negative rods, the expected antibiotic resistance phenotype was caps, vanr, tets, strs, penr, gens. But many isolates showed multidrug resistance including resistance to vancomycin, tetracycline, and streptomycin. From 248, 34 bacterial isolates (28 from field fish and 6 from hatchery-raised fish) that deviated from expected phenotype were selected to test for the presence of plasmid. Only three bacterial isolates (one from field fish and two from hatchery-raised) were found to have plasmids. This data suggests that antibiotic resistance in vast majority of isolates is of chromosomal origin.

After performing series of tests three isolates containing plasmids were identified as Serratia marcescens from Matson ditch creek chub; Peudomonas alcaligens or P. pseudoalaligens and Aeromonas salmonicida subsp smithia from tilapia. At this point we don’t know whether the plasmids found in these three isolates are responsible for conferring antibiotic resistance to these microbes, future studies are necessary. Furthermore studies to determine the source of antibiotic resistant bacteria are also needed.

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