Lecture 4: Biofilms Flashcards
Whats a biofilm
Community of microbes attached to a surface (biotic or abiotic) encased in a matrix
Biofilms are usually…
Multispecies
Can most bacteria form a biofilm
Yes under certain conditions
What are most water bacteria like
Not free floating organisms but rather grow upon submerged surfaces
Biofilm developement cycle
Mature biofilm
Planktonic cells detach
Motility, adhesion and environmental signals
Acyl-HSL signal reatach to mature biofilm - autoinducer quorum sensoring
Why form a biofilm
- Slow growth = Less energy output (less energy required)
- Nutrients are associated with surfaces
- Cooperation within community
- Provides protection from antimicrobial agents, predators and the immune system
Can bacteria choose to be in biofilm, is it always more favourable to be in a biofilm
yes and no there are times when bacteria WANT to detach
Where are Biofilms found?
What tends to be studied more (in bold)
Environment
**Industry
Medecine**
WHats the dif between a planktonic cell and a biofilm cell
- Hypothesis
- Approaches to address this hypothesis
Surface attached cells (biofilm) express dif genes compared to free-swimming (planktonic) cells
DNA microarray analysis
2D gel protein analysis
What did DNA array analysis determine
FOR biofilm cells:
Activated genes incluedd ones involved in protein translation and certain** types of metabolism**
Repressed genes included ones involved in** flagellar motility and secretion**
2D gel electrophoresis
- How to do it
- What it permited to determine (specificly what u have to remember from that slide)
Revise before exam
HOW:
- Seperate proteins by charge
- Seperate proteins by size
WHAT:
- 15 up regulated genes: one of which is pilA (pili-surface movement)
- 30 down regulated genes: one of which is fliC (flagellum-movement through liquid)
After manys studies of DNA microarrays and protein expression of planktonic cells and cells in biofilm, what is the consensus?
Planktonic cells and cells in biofilms EXPRESS different genes
No genes identified that are ONLY expressed in biofilms, in all bacteria
SO no biofilm markers have been identified
THis is because they’re are different strains and different growth conditions
What is an operunistic gram negative, rod bacteria that has a sequenced genome, is genetically amenable and best studied model for biofilms?
P.aeruginosa
aer ugi
nosa
What are features and advantages of pseudomonas aeruginosa
Features
- gram neg
- motile rod
- Pathogen of immunocomprimised, burn and CF patients
Advantages
- Sequenced genome
- Genetically amenable (i think this means like easily modifiable, easy to work with, easy to study)
- Best studied model for biofilms
Genes that are important for biofilm formation by P.earuginosa (dont name the genes, just talk about steps of formation and relation with gene expression)
1) *Platonic cells will reversibly attach to a surface (certain genes help with this)
2) *These cells with the express of another gene will irreversible attach
- = Monolayer formation
3) Other gene expression will permit microcolony formation
4) Other gene expression permits macrocolony formation (some planktonic cells can leave here)
How were genes imporant for biofilm formation identified
- Who
- Procedure used
WHO:
O’toole and Kotler screen
PROCEDURE USED:
- Random transpon-inserted mutant library
- Screened 2400 mutants
- Identified mutants that did not form biofilms and determined which genes were affected in the mutants
Random transposon-insertion mutant library how
Under specific conditions, transpons inserted randomly and one one time into the WT chromosome of P.aeruginosa
Amounf thousands of these mutants you should have an insertion in EACH non-essential gene
What procedure was used to test formation of biofilm and how was it done
Revise before exam
The 96-well microtitre plate assay
Cells innauculated into wells with dye that turns purple when there is biofilm formation. WT is the positive control (so should turn purple)
The mutants pilB and flgK did not turn purple (NEED TO KNOW THESE MUTANTS)
Review
Limitation of the 96 well procedure and what procedure is used instead (explain this other procedure)
YOu cant tell what stage of biofilm formation is formed in mutants
so we use Schematic of Flow cell system:
Uses a reservoir. A plum will pump liquid into flow cells which are cylinder tubes each containing different mutants (pilA, flgA and WT). Right before these tubes is an inoculation site. THen at the end. Waste
Biofilm sticks to glass, observe changes over time. Results:
- WT: Macrocolony formation after 8 hours
- pilB: microcolony formation, but nothing more
- flgK: Bacteria cant lach on to each other, no biofilm formation
Flagellum
flgK encodes a protein that is part of the flagellum (flagella is plural)
Important for swimming motility and/or attachement to surfaces
Pili
pilB encodes for a protein important for synthesis of type 4 PILI
Pili:
- Short, thin proteinaceous fibers
- cover entire surface of the cell
- Required for twitching motility (tests out where conditions are good, helps form macrocolonies to find new cells)
What do flgK and pilB usually help with
Revise before exam
flgK helps reversible attachemetn to surface: flagella
pilB helps to stick to other cells to form microcolonies: pili 4
What type of bacteria are gram negative rod, cause diarrhea, dysentery, kidney failure, bladder infections and pneumonia, virulence factors vary with strain, most strains are non-pathogenic
E.coli
Descibe E.coli
- Gram neg rod
- Causes diarrhea, dysentery, kindey failure, bladder infections and pneumonia
- Virulence factors vary with strain (1 that causes diarrhea will not cause a urinary tract infection)
- Most strains are non-pathogenic (live in our gastrointestinal tract)
- Uropathogenic E.Coli (UPEC) causes urinary tract infections
Names of strcutures that help with E.coli biofilm formation
Revise before exam
1) Flagella: helps with initial attachment
2) Type I pili and Ag43 help with attachment to form monolayer
3) Flageela helps to form microcolony
4) Colanic acid helps to form macrocolony
Properties of mature biofilm?
- Surrounded by exopolysaccharide matrix
- Heterogeneity-bacteria throughoout the biofilm experience different environments
- Resistant to antimicrobial agents
Function of matrix and how do we know this
- Structure
- Protection
HOW:
- Observation under microscope
- Identification of important elements of the matrix and then deletion of important genes to determine what their individual functions are
Matrix composition
Poysaccharides
DNA (eDNA for extracellular DNA)
Proteins
Polysaccharides in the P.aerurginosa
Revise before exam
- Alginate
+overproduction occurs in CF patients - PsI
+Mannose and galactose rich polysaccharides - pel
+Involved in pellicle formation (floating biofilm)
+Glucose rich polysaccaride
eDNA
- Where does it come from
- Whats its function
Secreted by cells in the biofilm (active process, not just cuz cells die)
Function:
- Strucutre
- Protection of the cell in the biofilm (more in antibiotic resistance lecture next week)
Heterpgeneity
- What causes it
- Why does it matte
dif environment depending where bacteria is located in bacteria:
- Waste (builds up deep in biofilm
- Nutrients(more on outside, less inside)
- Signaling molecules (cells produce this)
- Antibiotics
It matters because different genes are expressed depending on location but only slight differences
Planktonic antibiotic resistance resistance mechanisms
Intrinsic (part of genome)
- Low outer membrane permeability
- Action of efflux pumps
Acquired (through mutation)
- Upregulation of drug efflux pumps
- Alternation of antibiotic target
Biofilm-specific resistance mechanisms
- Multiple mechanisms
- Not based on mutations
- Matrix (eDNA)
- Biofilm-specific gene expression
Beneficial Biofilms
In:
- Water treatment
- Energy production (microbial fuel cells)
- Nitrogen fication
- Biocontrol (protecing crops)
Wastewater treatment
Primary, secondary and tertiary
In the aeration tanks, biofilm and planktonic bacteria break down organic matter (secondary)
Microbial Fuel Cells (MFCs)
Harness energy potential and water generated when bacteria metabolises substate
COnsist of anode, cathode and a proton or cation exchange membrane and an electrical circuit
Electrical power is generated because of potential difference between anode and cathode and because of the flow of electrons
Point of this: Biofilmas can be benifcial
Plants and biofilms
Alfama and soybean fix nitrogen
p.fluorescens protect tomatoes from rot
Nitrogen fixation
- Nitrogen is required by all living organisms (proteins, nucleic acids)
- Earth’s atmosphere is arround 80% nitrogen gas (unusable)
- Must be fixed: reduced (combined with H) to ammonia by nitrogen fixing bacteria
- Green plants use the fixed nitrogen to produce proteins that are then passed onto the food chain
Examples of plants and bacteria positive ineractions
S. melitoti and clover where nitrogen fixation occurs
**BIOCONTROL: **Fungus rots tomatoes so p.Fluorescens biofilm coat tomatio root to protect the tomato from pathogens
Problematoc biofilms exmaples
In industry:
- Clogging and corroding pipes
- Marine biofouling (on ships)
In environment:
- plant pathogens
In medecine
-65% of human bacterial infections are biofilm based
Biofilms in Medecin
- 65% of human infections are biofilmed based
- Biofilms can form on anything you put in your body or on tissue in your body, they can detach and attach to other things as well
- Biofilm-based infections marked by recurring symptoms after each use of antibiotics (cuz they can be 10 to 1000 times more resistant to antibiotics compared to planktonic counterparts)
Examples of diseases caused by biofilms in medecine
Recurrent urinary tract infections by E.coli
CF by p. aeruginosa
A model of biofilm development
1) Mature biofilm
2) Detachment signals
3) Planktonic cells
4) Planktonik cells get environmental cues and initial interaction happends
5) Quorum signals
6) Attaches to mature biofilm
When are bacteria harmful throughout biofilm stages
and why are biofilm cells recurring natured
When cell is in mature biofilm it does not express virulence factors so the biofilm acts as a reservoir
Planktonic cells cause symptoms of acute indections and so express virulence factors like toxins
Antibiotics kill the planktonic cells but not the biofilm cells. this is where the recurring nature of biofilm based infections comes in
Chronic vs acute infections
actue: plaktonic cells expressing virulence factors
Chronic (long lasting): Don not express virulence factors
Whats focused on when studying biofilms and diseases
Providing evidence that the infection is BIOFILM based the to identify approaches to eliminate the biofilm
What is CF
Revise before exam
- Most common fatal genetic disease to affect young canadians
- cuz of mutation in CFTR
- Chronic bacterial infection in their lungs that persist even if extensive antimicrobial therapy is used (therapy helps to have stable periods, exacerbations is when things get bad)
What evidence is there for biofilm in the CF lung
Based in quorum-sensing autoinducer profiles (indicative of biofilm growth as opposed to planktinik growth)
Microscopy
EXPLAIN
Quorum sensing: A bacterial cell density sensing system
- Each cell produces an autoinducer (autoinducer is used as a measure of population density)
- As the population density increases, so does the concentration of the autoinducer
- At some point autoinducer conc reaches a CRITICAL THRESHOLD and binding of autoinducer to receptor occurs
- THe receptor goes on to interact with DNA affect gene expression
Relationship autoincucer and p.aeruginosa
in this bacteria there are 2 dif autoinducer types and THEIR RATIO DEFFER depending on whether they are growing on:
- Planktonik cells
or
-Biofilms
How does p.aeruginosa biofilm in CF lung persist
Virulence factor type III secretion system that injects proteins into host cells is not expressed when in biofilm
How does p.aeruginosa cause damage in CF lung?
INDIRECT damage done by biofilm:
- Immune cells are recruited but cant kill biofilm infection
- Protease, free radicals produced by immune system damages lung tissue
What is the CF lung environment and how is this promising
Thick mucous
Low oxygen
allowung biofilm formation
hypothesis is that p.aeruginosa biofilms will behave differently when grown anaerobically and maube this will result in a novel approach for therapy
p.aeruginosa biofilms aerobic and anaerobic conditions how it is studied
Anaerbobic: biofilm growth otherwise no
2D gel analysis spot 29 is present in aerobic biofilm but not in aerobic and this is OprF (porin)
oprF importance for biofilm formation in P.aeruginosa
Very little biofilm formation when it is not present
How do mutations accumulate over time of P.aeruginosa in CF lung
Slow growth = stressful conditions = accumulation of mutations in the bacteria
What mutations accumulate over time in
as P.aeruginosa adapts in CF lung
Revise before exam
mucA = results in mucoidy, increases production of alginate because mucA is a a negative regulator of alginate production. alginate attenuates inflammatory respons so helps to avoid detection by immune response. Apperance of mucoid strain correlates with poor clinical outcome
mexZ = MexZ protein is a negative regulator of MexXY that codes for efflex pump that increase resistance to aminoglycosides (an antibiotic)
UTI’s
- Considered to be 1 of the most COMMON bacterial infections
- Tends to recur despite antibiotic therapy
- Most common isolate is uropathogenic E.coli (UPEC)
Experiment used to proove that UTI’s are biofilm based infections
Used mice. DId z-series to show bacteria are located thoughout the pod and biofilm is formed
Biofilm formation Ecoli, important genes and role
Type I pili and Ag43 are impotant for E.coli to attach to surface
control of biofilm formation
You need to identify the genes important for biofilm formation and develop approach to prevent their function
1) O’toole and Kotler screen for biofilm deficient mutants
2) Educated guessing:
- Quorum sensing in cell density system that is important for many communitu based behaviour
- so QS must be important for biofilm formulation
2 major QS systems in p.aeruginosa
Revise before exam
1) Las:
- Las R is a repressor of lasI gene that codes for lasI that forms autoinducer. autoinducer represses las R
2) Rh1
- RhIR is the repressor of RhII gene that codes for RHII that forms autoinducer that represses RhIR
Is QS involved in biofilm formation?
a lasI mutation affects biofilm formation:
- WT: macrocolony
- lasI mutant: no macrocolony
- LasI mutatn + autoinducer: macrocolony
QS inhibitor
Revise before exam
A QSI and D. pulchra is an australian sea weed that does this: it forms furanone 2 which is the real QSI
Observation: no bacteria growth on this seaweed
so it must have an anti-biofilm compound
What are some QS controlled processes
Biofilm formation
Fruiting body formation
Production of antibiotics
Expression of virulence factors
Expression of bioluminescence
Motility
What techinc was used to figure out how the seaweed QSI worked, like what process it shut dows
Assay system:
A bacteria that does swarming is basicly a community based motility controled by QS. SO swarm cells move and inner cells grow which forms this colony
You add graudyally more and more of this sea weed in each different fraction and see effects. the sea weed forms furanone which is the QSI that distrupts quorum sensing because swarming decreases at every fraction that you higher FURANONE 2 concentration
How are biofilms studied
In vivo real hard: if so patient samples, environmental biofilm samples
Most labs in vitro and monospecies
- Growth media can mimic in vivo
- some labs use other species and mix them together
How can you detect a biofilm
1) simply count bacteria: vortex, scrape, sonificate cells off a surface and grow them up on nutrient plates
2) Visualize the bacteria: label with fluoresence
Biofilm detachement
Natural part of biofilm lifecycle and get be released in clumps or single cells
Identification of a detachement factor
p.aeruginosa will detach once stop of fresh nutrient flow has stopped
if you add spent media (bacteria media that has all its nutrients sucked out of it) on to biofilm there will be detachement and monolayer will be formed
Some bavteria kill biofilms
