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There Are Three Main Mechanisms of Action That Antibacterial Drugs Use PREVALENCE OF ANTIBIOTIC RESISTANT COMMENSAL BACTERIA IN ENDANGERED AVIAN SPECIES by Caroline Ann Efstathion A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Science in Partial Fulfillment of the Requirement for the Degree of Master of Science Florida Atlantic University Boca Raton, FL May 2011 PREYALENCE OF ANTIBIOTIC RESISTANT COMMENSAL BACTERIA IN ENDANGERED AVIAN SPECIES by Caroline Ann Efstathion This thesis was prepared under the direction of the candidate's thesis advisor, Dr. wadiuto Esiobu, Department of Biological Sciences, and has been approved by the members of her supervisory committee. It was submitted to the faculty ofthe Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree ofMaster ofScience. .dt College ofScience 4n'/ /5"t~/l Date III ACKNOWLEDGEMENTS The author wishes to thank Dr. Nwadituo Esiobu for the opportunity to work with her and for all her hard work and dedication to this study. The author also wishes to thank Rare Species Conservatory Foundation, Voren Aviaries, and Backos Bird Clinic for their assistance in conducting this study. The author is grateful for finical assistance provided by The Treasure Coast Exotic Bird Club of Stuart Florida. iii ABSTRACT Author: Caroline Efstathion Title: Prevalence of Antibiotic Resistant Commensal Bacteria in Endangered Avian Species Institution: Florida Atlantic University Thesis Advisor: Dr. Nwadiuto Esiobu Degree: Master of Science Year: 2011 This study examines prevalence of antibiotic resistant bacteria in Amazon parrots in three different groups, one wild and two captive. Commensal bacteria were isolated from 24 parrots and screened for antibiotic resistance. Culture-based and molecular methods were used and a total of 546 isolates from the genus Staphylococcus and Rothia were obtained. Antibiotic resistance was found in all parrot groups in 161 isolates with 48 having multi-drug resistance. The highest frequency of resistance was found to Enrofloxacin and Amoxicillin/Clavulnate though all antibiotics tested were resisted by some bacterial isolates. Both captive groups exhibited more resistant individuals than the wild. This study concluded that antibiotic resistant commensal bacteria in Amazons are common; however, patterns found cannot be explained by antibiotic use. A high rate of multi-drug resistance was detected in more common mannitol non-fermenting Staphylococcus spp., and drug resistant detected in Rothia spp. may indicate a future role in disease. iv DEDICATION This manuscript is dedicated to my parents and Ron. I also dedicate this work to my late birds, Moondust, Cricket, and Lulu, whom started me down the path that led to this work. THE PREVALENCE OF ANTIBIOTIC RESISTANT COMMENSAL BACTERIA IN ENDANGERED AVIAN SPECIES TABLES ............................................................................................................................... viii FIGURES ................................................................................................................................. ix 1.0 INTRODUCTION .......................................................................................................... 1 1.1 Antibiotic agents .................................................................................................... 1 1.1.1 Resistance Mechanisms against Antibiotics ........................................... 2 1.2 Commensal Bacteria in Spread of Resistance ..................................................... 2 1.3 Antibiotic use in non-clinical areas ...................................................................... 3 1.4 Antibiotic Resistance in Conservation Medicine ................................................ 4 1.5 Commensal Bacterial Associated with Antibiotic Resistance and Possible Consequences to Human Health ........................................................................... 4 1.6 Prevalence of Selected Commensal Bacteria in Parrots ..................................... 5 1.7 Study Aims............................................................................................................. 6 2.0 MATERIALS AND METHODS................................................................................... 7 2.1 Antibiotics – Mechanisms of Action and Rationale for Selection ..................... 7 2.2 Amazon Parrot Groups Sampled .......................................................................... 8 2.2.1 Sampling of Amazon Parrots ................................................................. 10 2.3 Microbiological Media Employed...................................................................... 11 2.3.1 Mannitol Salt Agar for Isolation of Staphylococcus species................ 11 2.3.2 MacConkey Agar for Isolation of Escherichia coli .............................. 11 2.3.3 Bile Esculin Azide Agar for Isolation of Enterococci .......................... 11 v 2.4 Isolation, Characterization, and Identification of Bacterial Commensal Isolates.................................................................................................................. 12 2.4.1 Spread Plating for Isolation of Bacterial Commensals ......................... 12 2.4.2 Initial Characterization of Bacterial Commensal Isolates .................... 12 2.4.3 Molecular Identification of Commensal Bacteria ................................. 13 2.4.4 Sequence Analysis .................................................................................. 14 2.5 Determination of Antibiotic Resistance Isolates ............................................... 14 2.5.1 Media Preparation for Agar Dilution ..................................................... 14 2.5.2 Screening for Antibiotic Resistance ...................................................... 14 2.5.3 Determination of Minimum Inhibitory Concentrations of Representative Isolates ........................................................................... 15 2.6 Statistical Analysis .............................................................................................. 16 3.0 RESULTS ...................................................................................................................... 17 3.1 Isolates Obtained from Amazon Groups Sampled ............................................ 17 3.2 Prevalence of Target Commensal Bacteria ........................................................ 17 3.2.1 Enterococci .............................................................................................. 17 3.2.2 Gram negative coliform bacteria............................................................ 17 3.2.3 Escherichia coli ....................................................................................... 17 3.2.4 Staphylococci and other Catalase and Gram Positive Cocci................ 18 3.3 Identification of Gram Positive Cocci Isolated from Amazon Parrots ............ 18 3.3.1 Operational Taxonomic Units for Isolates Obtained from Mannitol Salt Agar Plates ....................................................................................... 18 3.3.2 Molecular Identification of Gram positive cocci .................................. 18 3.3.3 Determination of Bacterial Species ....................................................... 19 3.4 Prevalence of Antimicrobial Resistance ............................................................ 19 vi 3.4.1 Antimicrobial Resistance of the Gram positive cocci from all Groups of Amazon Parrots ..................................................................... 21 3.4.2 Comparison of Antibiotic Resistance among Bacterial Species .......... 21 3.4.3 Minimum Inhibitory Concentration for Representative Isolates ......... 21 3.5 Statistical Analysis of Isolates ............................................................................ 22 3.5.1 Analysis Among Isolates from the Amazon Groups ............................ 22 3.5.2 Analysis Among Bacterial OTU ............................................................ 22 3.6 Individual Parrot Results ..................................................................................... 22 4.0 DISCUSSION ............................................................................................................... 24 4.1 Commensal Bacteria Isolated from Amazon Parrots ........................................ 24 4.1.1 Incidence of E. coli and Gram negative Coliform Bacteria ................. 24 4.1.2 Gram-Positive Cocci Species Isolated ................................................... 25 4.2 Antibiotic Resistance in Amazon Parrots .......................................................... 27 4.3 Clonal Nature of Bacteria ................................................................................... 27 4.4 Antibiotic Resistance found in Wild Birds ........................................................ 28 4.5 Birds as Reservoirs and Vectors for Antibiotic Resistant Bacteria .................. 30 4.6 Importance of Enrofloxacin and Amoxicillin/Clavulnate Resistance .............. 31 4.7 Conclusion ........................................................................................................... 31 REFERENCES ....................................................................................................................... 43 vii TABLES Table 1: Details of Individual Parrots Sampled ............................................................... 33 Table 2: Antibiotics and concentrations used to screen isolates obtained from choanal and cloacal samples of Amazon
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