Abstract

Staphylococcus aureus chromosomal DNA from 125 methicillin-resistant and 10 methicillin-sensitive clinical isolates was digested with rare-cutting restriction endonucleases and subjected to pulsed-field gel electrophoresis (PFGE). Thirty-nine distinct genomic macrorestriction patterns (GPs) were identified using SmaI; 29 of these patterns are from methicillin-resistant S. aureus (MRSA).

A dendrogram showing percent similarity among the patterns was constructed which revealed the considerable genomic diversity of the collection even though the isolates had been obtained during outbreaks. The majority of the MRSA formed one broad group with two outbreak subclasses. However, 8 of the 29 MRSA GPs (28%) were diverse.

Digestion with CspI, which clarified relationships among some SmaI patterns, showed that 5 strains had much larger genomes (3.5 to 3.8 Mb) than the average of our isolates (3.0 Mb). Duplications of large regions of their chromosomes may have occurred.

Restriction fragment length polymorphisms (RFLPs) of the methicillin resistance gene, mecA, and the transposon, Tn554, were used to further type selected isolates. The association of the same mecA and Tn554 RFLPs with diverse MRSA indicates that mecA may have been horizontally transferred to diverse S. aureus strains on multiple occasions.

In the process of examining genomic differences among the isolates, aberrantly migrating bands were observed. Evidence that these bands were supercoiled plasmids greater than 300 kb was obtained by linearizing the plasmids with S1 nuclease and determining their sizes with PFGE. The "S1-PFGE" method was developed in our laboratory as a general means of identifying and sizing very large plasmids that often escape detection.

The SmaI genomic polymorphisms of two related isolates were attributed to the insertion of temperate bacteriophages both of which lacked SmaI sites. DNA of one of these isolates contained an additional SmaI site possibly created by point mutation.

One MRSA isolate was serially propagated in antibiotic-free medium for 29 days. The serial culture showed a decline in the percentage of MRSA cells. PFGE analysis showed that the mecA gene had been deleted. Together, these observations indicate that S. aureus, particularly MRSA, may be more diverse than has been appreciated in the past.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology, Molecular Biology and Biochemistry

School

Graduate School

First Advisor

Anthony J. Zuccarelli

Second Advisor

Barry L. Taylor

Third Advisor

Junichi Ryu

Fourth Advisor

David Hessinger

Fifth Advisor

Brad Hyman

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1997

Date (Title Page)

6-1997

Language

English

Library of Congress/MESH Subject Headings

Staphylococcus aureus -- genetics; Variation -- genetics; Staphylococcus Phages -- genetics; Genes, Bacterial.

Type

Dissertation

Page Count

ix; 179

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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