Lecture 5 - Biofilms

Question 1

Describe how bacteria arrive at a surface to form a biofilm

Answer 1

Arrival of bacteria to a surface
Non-motile bacteria can reach a surface via diffusion (however turbulence impaction can make this more likely) and motile bacteria can use chemotaxis to direct movement towards the surface.
Between the surface and the flow of fluid/air there is a boundary layer where there is no flow.
Laminar flow
* Non-motile bacteria in the layer/s of laminar flow closest to the surface can then diffuse through the boundary layer to the surface
* Motile bacteria can move from layers of the laminar flow further from the surface via chemotaxis
Turbulent flow
In turbulent flow there is lots of mixing and the boundary layer is disturbed meaning it is smaller. The liquid can also hit the surface. This increases the probability of bacteria reaching the surface.
The turbulence is increased when the surface is rough which increases the probability further

Question 2

Why are attachment protiens important in bacteria.

Answer 2

Attachment of bacteria to a surface
When a bacteria arrives at the surface there is a repulsion due to the mutual negative charge of the bacteria and surface (Brownian movement).
In a lab this can be overcome by drying. In nature bridging structures help attachment. (generic adhesions: flagella, Pilli, fimbriae, stalks, (lipo)teichoic acids etc.)

Question 3

What are generic adhesins

Answer 3

Generic adhesins (encoded in nucleoid)
Generic adhesins can bind non-specifically by electrostatic forces: it requires abiotic surfaces and conditioning film.
They can also bind specifically mainly via glycoprotein receptors (all pathogenic bacteria can do this as well as some non-pathogenic bacteria)
Reversible attachment

* Generic adhesins (all bacteria)
	○ Flagella - binds to toll-like receptor 5 (not a glycoprotein)
	○ Fibronectin - glycoprotein mannose
	○ Teichoic and lipoteichoic acid
* Generic adhesins (pathogens)
Adhesins of pathogens specifically bind to sugars in specific glycoprotein receptors (tissue specific)
	○ Pili
	○ Fimbriae
* Extra adhesins - pathogens only (often contained within plasmid)
Generic adhesins aid initial attachment only
Pathogens must dock with membrane adhesion proteins using attachment proteins.

Question 4

What are attachment protiens?

Answer 4

Attachment proteins
Attachment proteins dock the bacterial cell onto the host cell. They are sugar binding proteins and are encoded in plasmids.
They bind to sugar residues in host receptors (adhesion proteins)
* Glycans
* Glycoproteins and glycolipids (mainly)

Gram -ve attachment proteins (Trimeric Autotransporter Adhesins TAAs) also known as Type IV secretion system.
Each specific pathogen has a specific receptor.

Gram +ve attachment proteins -
e.g. Group A streptococci - Protein M
as each pathogen has a specific receptor the best way to stop disease is to prevent attachment by blocking receptors.

Question 5

What is irriversible attachment in regards to biofilm formation.

Answer 5

Irreversible attachment
A phenotypic switch occurring when enough bacteria are present leads to exopolymer deposition which cements the bacteria in place.
Once cells attach to a surface they form clusters (helped by generic adhesins). When enough cells are present, they communicate with each other and upregulate certain genes
One important gene is alg - for alginate production.
* Over secrete alginate to form a slime
* Cements cells to surface
* Biofilm now begins to grow via in-house division

Question 6

How do baverial cells comunicate.

Answer 6

The cells need to communicate to do this they use cell signalling. Signalling molecules include: (autoinducers)
* Gram-positive cells: oligopeptides
* Gram-negative cells: acyl-homoserine lactones (AHL, HSL) - have lactone ring in common.

Cells always produce signalling molecules but with too few cells are dilute so don’t reach each other, this means there is no co-ordinated response until the density of cells increases and the threshold is reached.

LuxIR regulon - Aliivibrio fischeri
Regulatory Gene Lux I codes for HSL
Lux R is the activator protein
At low density the Lux I produces HSL however this is lost in the void. The HSL conc is too low to activate Lux R.
At high density Lux I produces HSL, the overall HSL conc is high so it re-enters the cell and Lux R is activated causing the genes encoding light production are expressed.

Other lux-type regulons
When concentration is high it causes the upregulation of beneficial genes and down regulation of genes that are no longer necessary such as those coding for flagella
LuxI/LuxR homologues are found in many genera and species

Question 7

Describe mature biofilms

Answer 7

Mature Biofilms
Biofilms are very hydrated containing approx. 98% water. The water contains nutrients, waste, etc. and exopolymeric substances (forms the matrix framework providing structure and integrity).
The first bacteria that attach divide and provide more matrix. Within the layers of matrix there is a disproportionate level of nutrients. The bacterial growth rates and EPS production is greater in layers closer to fresh nutrients.
This leads to the formation of structures known as mushroom stacks.
The stalk of the mushroom is thinner and contains gaps where liquid can pass through.
The topography of the biofilm can change depending on flow rate and health.

Question 8

How are bacteria detached.

Answer 8

Sloughing
* Turbulence
* Scouring
* Grazing - phagocytes
Programmed detachment
* Synchronised liberation of hydrophilic daughter cells which quickly leave the biofilm. It takes 2-3 divisions before attachment is possible.

Biofilms are beneficial and safe for bacteria and are therefore much harder to kill