Bacteriology

• how do bacteria 'count their numbers'?

  quorum sensing
• name an example of a bacteria that does quorum sensing;: v. fischeri has Lux1, which sends out autoinducers, and LuxR, which detects them. LuxR then turns on the LuxCDABE genes, which causes bioluminescence.
• what are gram- autoinducers?

  acetylated homoserine lactones, AHLs
• how do different AHLs distinguish themselves? is all the same repeating molecule, but the side chain differs in length and modifications
• what are gram+ autoinducers?

  (oligo)peptides
• what is the main difference between gram- and gram+ autoinducers? AHLs are lipids, and can diffuse over the membrane. peptides are small, but do not cross the membrane and therefore need to be actively transported both in an out.
• what is AI-2? the interspecies oligopeptide autoinducer, which counts all total bacteria present
• which receptors are associated with AI-2? LuxP --> LuxQ --> LucU --> target genes
• what are the three steps of biofilm formation? 1. initial adherence

  2. microcolony formation

  3. biofilm maturation + dispersion
• what is up- and downregulated during biofilm formation step: adherence? + CWA proteins (for adhesion)

  - virulence factors
• what is up- and downregulated during biofilm formation step: microcolony formation? + EPS (extracellular polymeric substance)

  + autolysins
• what is up- and downregulated during biofilm formation step: biofilm maturation? + dispersion enzymes

  + virulence factors
• define biofilm;: organized microbial communities consisting of layers of cells associated with surfaces and surrounded by EPM
• name an example of a gram+ and gram- biofilm-forming bacterium;: + s. aureus

  - p. aeruginosa
• what does EPS consist of? - proteins

  - eDNA

  - polysaccharides
• how can you determine the age of a biofilm? at the start the EPS is mostly eDNA, the percentage of eDNA helps determine the age
• what are the survival benefits of forming biofilm? - difficult to penetrate for immune cells

  - different diffusion rate prevents antibiotics/AMPs from entering

  - secrete immune evasion molecules

  - target immune response to EPS instead of bacteria
• how do you treat biofilm? - mechanical removal

  - long term antibiotics (>3 months)

  - replace affected implant
• how do you study biofilms? - 96 wells plate crystal violet assay

  - microscopy

  - flow cell
• how does a 96 wells plate crystal violet assay work? crystal violet binds everything attached to the well. add EPS degrading enzymes (DNAses, proteinases), and measure the OD595.