AN INVESTIGATION INTO THE CORRELATION BETWEEN SPECIFIC GENETIC
MUTATIONS AND COLISTIN RESISTANCE IN ESCHERICHIA COLI.
Presenter: Ms. Leisha McGrath, College: Galway Mayo Institute of Technology
Prior to the discovery of the plasmid-borne mcr-1 gene (1), resistance to the antibiotic colisitin was limited to chromosomal mechanisms. The emergence of mcr-1 implies resistance to this last resort antibiotic can now disseminate rapidly. However, research at UCD’s Centre for Food Safety revealed that colistinresistant Escherichia coli isolates lacking mcr-1 are more resistant to colistin compared to mcr-1 positive isolates.
To better understand chromosomally-mediated colistin resistance versus plasmid-mediated resistance, the chromosomal gene phoP was deleted from an E. coli ATCC 25922 control strain. Deletion of phoP, a regulatory gene associated with bacterial chromosomal colistin resistance, was achieved by Lambda Red Recombination (2). This method involved restriction enzyme excision of phoP, replacing it with an antibiotic resistance cassette which was then subsequently removed. This created a phoP negative mutant control strain. The minimum inhibitory concentration (MIC) of the wild type and mutant control strains, as well as six test E. coli isolates, to colistin was then investigated using microbroth dilution.
Successful deletion of phoP from the mutant strain was demonstrated by polymerase chain reaction amplification of the target gene in both the wild type and mutant strains, and electrophoresis of the amplicons. The MIC studies illustrated a significant one-fold increase in the MIC of colistin from the wild type to the mutant control strain (0.125mg/l versus 0.25mg/l). Furthermore, colistin-resistant E. coli test isolates lacking mcr-1 exhibited a colistin MIC double that of colistin-resistant test isolates with mcr-1 (8mg/l versus 4mg/l).
The one-fold increase in the MIC of colistin in the mutant E. coli would indicate that phoP alone cannot mediate colistin resistance. Instead, a repertoire of chromosomal genes regulating colistin resistance are required for strongly resistant phenotypes. Similarly, it could be suggested that mcr-1 alone does not confer high colistin resistance – rather, it seemingly augments chromosomal resistance mechanisms already present.
AN INVESTIGATION OF THE EFFECT OF SFV INFECTION ON P32 LEVELS
AND DISTRIBUTION IN BHK CELLS.
Presenter: Mr. Liam A. Rayman
College: Cork Institute of Technology/ University College Cork
The cellular protein p32 has been shown to have many possible functions within the cell. It is believed that one of these functions involves binding to the capsid protein of Semliki Forest Virus (SFV); an interaction that may provide a greater understanding of both SFV replication and p32 involvement in the cell.
Here we show that SFV infection affects levels of p32 and its distribution within the cell. A cell fractionation procedure, known as the Rapid, Efficient And Practical (REAP) method, was utilised to separate the cytosolic components from the nuclear components. From these fractions it was established, employing Western Blot analysis, that SFV has a significant effect on p32 altering its distribution between the nucleus and the cytosol of infected BHK-21 cells. Additionally, examination of viral lysates revealed that p32 is present in SFV mutant virions .The interaction between the SFV capsid protein and p32 was determined to be discrete from the interaction between the Rubella capsid protein and p32.
The results of this study demonstrate that while much is still to be understood about p32, it has an important role to play in SFV infection in a role that is only now being uncovered.