Development of a restriction endonuclease analysis method for differentiating human and animal derived faecal streptococci : A thesis submitted in fulfilment of the requirements for the Degree of Master of Applied Science in Lincoln University

Abstract

One hundred and seventy-three group D streptococci were isolated from the faeces of humans and cows, and presumptively speciated using a battery of biochemical and physiological tests. A minimethod for extracting chromosomal DNA from these organisms was developed, based on the larger scale method of Rodriguez and Tait (1983) for extracting Bacillus subtilis chromosomal DNA. Problems were experienced in obtaining a method which would consistently produce DNA pure enough for cleavage by restriction endonucleases; however, addition of extra ethanol precipitation steps to the end of the method overcame this. Chromosomal DNA from 126 human enterococci and 10 bovine group D isolates was subjected to restriction endonuclease analysis (REA) using the enzyme EcoR1. It was hoped that examination of band patterns produced by REA would reveal differences between isolates that could be related to their faecal source - human or bovine. Isolate REA "fingerprints" were so diverse, however, that it was not possible to do this. Fingerprints common to different isolates from the same host individual were seen, but fingerprints common to isolates from different human or bovine individuals were not observed. A preliminary survey of the plasmid content of 47 human enterococci isolates, as well as group D streptococcal type culture strains, was also carried out. 85% of the human isolates contained plasmids, and it appeared that some isolates may have shared the same plasmid(s).