Date of Award

6-2011

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Michael P. Shiaris

Second Advisor

Rachel C. Skvirsky

Third Advisor

Katherine E. Gibson

Abstract

Vancomycin is a last-line glycopeptide antibiotic used to treat Gram-positive bacterial infection. Enterococcus, as the third most common bacterial cause of nosocomial infections in the United States, has evolved transferable vancomycin-resistant genes such as vanA and vanB. High numbers of Enterococcus have been detected in Wollaston Beach, Quincy, MA. The beach is influenced by several storm drain outlets that are contaminated by sewage from the city of Quincy, as well as the contaminated Furnace Brook that originates in the Blue Hill Reservation. A major objective of this work was to examine whether vancomycin-resistant Enterococcus (VRE) are present in Wollaston Beach sands. Culture methods, physiological tests and molecular approaches, including PCR and DNA sequencing, were used to detect VRE. The most probable number (MPN) method was used to quantify Enterococcus counts in the surface beach sands. High counts of Enterococcus (up to 6.20×103 MPN 100 cc-1 sand) were detected from three sample sites during the course of four months of sampling. Four vanA-positive VRE strains were recovered from Wollaston Beach sands, and the VRE counts were 205 MPN 100 cc-1 sand in one sample. This is the first report to quantify VRE numbers in the environment in the United States. VRE in sands can serve as a vancomycin resistance (VR) gene reservoir, and potentially a source of virulent genes, to other bacteria. In the process of enriching for Enterococcus isolation, an abundance of naturally-resistant VR Gram-positive bacteria (non-Enterococcus) were recovered from Wollaston Beach sands, suggesting the non-Enterococcus bacteria can compete with Enterococcus in vancomycin amended media. Both standard biochemical tests and Enterococcus-specific PCR misidentified non-Enterococcus species as Enterococcus. In summary, the presence of VRE in Wollaston Beach sand is common, and it indicates various contamination sources of the beach. A combination of different selective media and molecular methods are necessary to enrich and identify VRE from mixed bacterial community samples.

Comments

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