Using the counterselectable marker pheS' to study the excision rate and excision patterns of the pathogenicity island of Enterococcus faecalis V583

Sammanfattning: The Enterococcus genus consists of natural members of the gastrointestinal tract but they are also opportunistic pathogens. They are a common cause of urinary tract infections but can also cause sepsis and other infections. Enterococcus faecalis and Enterococcus faecium are the most abundant in clinical specimens. Enterococci are a leading cause of nosocomial infections and they have developed resistance against a number of antibiotics e.g. vancomycin. E. faecalis V583 was the first vancomycin resistant isolate reported in the U.S. Movable genetic elements such as pathogenicity islands, PAI, are important for bacterial evolution. PAI:s are large chromosomal fragments mostly seen in pathogenic strains and carry regions such as transposons and insertion elements along with virulence factors and transfer genes. A PAI has been detected in the chromosome of E. faecalis. Excision of PAI:s has been studied for uropathogenic E. coli and frequencies of 10-5 and 10-6 have been reported. In this study the excision rate and excision patterns of E. faecalis V583 was studied using the counterselectable marker pheS', causing p-Cl-phe sensitivity, and a chloramphenicol resistance gene, cat, inserted at two different positions of the PAI and selecting for excisions by growth on p-Cl-phe. Excision rates of 10-6 and 10-8 were seen based on the p-Cl-phe resistance and chloramphenicol sensitivity. Mutation rate in the pheS' gene was high compared to excision rate which made the method difficult to work with. No obvious excision patterns were detected but the excisions seemed to be limited to the close surroundings of the pheS'/cat insertion.