Today we had a guest speaker, Katie Wilcox, that came and spoke to our class. Here are some of the notes that I got out of her presentation over "Iraqibacter"
Microbiology
September 9, 2009
Guest Speaker: Katie Wilcox
Acinetobacter Baumannii
• Gram-negative bacillus
• Opportunistic pathogen
• “Iraqibacter”
• Inherently susceptible to few antibiotics; increasingly multi-drug resistant
• Bacteremia and ventilator-associated pneumonia
• 2 Noted Characteristics:
o Ability to grow in serum
o Ability to survive on abiotic surfaces and skin for extended periods of time
• Transposon mutant library
o 6000 mutants
o Screened for loss of ability to grow in serum and also for loss of ability to bind to polysterene 96-well plate
• Characterization of virulence factors:
o 6 mutants that grew in serum were identified
o PFGE
o DNA isolation
o Enzymatic digestion and ligation
o Inverse PCR (primers for Transposon)
• Confirm presence of product by gel electrophoresis
• Extraction of products from gel
o Ligation of inverse PCR product into pCR II vector
o Transformation into OneShot INVaF’ cells
• X-Gal – blue/white screening
o Plasmid purification
o Sequencing
• Gene Sequencing and RT-PCR
o Used BLAST online tool to analyze results
o Putative transcriptional regulator
• Target for antibiotic development?
o Further Investigation
• RT-PCR: uses reverse transcriptase; allows analysis of gene expression at different time points/under different conditions
• Different points in growth cycle
• GeneChip Studies
o Uses microarray technology to compare differential gene expression wild type to mutant
o Complicated process of RNA isolation, conversion to cDNA, labeling, hybridization to the chip, etc.
o Found ~30 genes that are differentially expressed in the mutant that inhibit it from growing in serum – Not actually sure what it’s doing to it.
• Lots of applications for future work
• Confirm results
• Learn the role of this transcriptional regulator in vivo
• Figure out if it would be a suitable target for an antibiotic
• Surface Binding Abilities
o Abiotic Surfaces (Polysterene) – Photos on slide show
o Fibrinogen – Photos on slide show
