Lecture 4 - Biofilms Flashcards
Biofilm is defined as a community of _____ attached to a ______ (biotic or abiotic), encased in a ________.
Most biofilms are __________.
_____ bacteria can form a biofilm under certain conditions.
microbes; surface; matrix.
multispecies
most
What are planktonic cells?
Free-swimming cells.
These cells can make biofilms when gathered as a community.
(T/F) Biofilms were observed in the lab first.
False.
Biofilms were observed in nature (freshwater) before the lab.
“it is quite evident that for the most part the water bacteria are not free floating organisms, but grow upon submerged surfaces”
Briefly describe the biofilm development cycle.
Planktonic cells utilize motility, adhesins, and environmental cues, followed by quorum sensing, to develop into mature biofilms. Some cells detach from the biofilm and return to a planktonic state.
Detached cells can remain planktonic or start a biofilm somewhere else.
Biofilms are ____-like structures.
These structures have ______ for liquids to flow through allowing for _________ and _______.
pillar
channels; nutrient intake; waste removal
Why form a biofilm?
1) slow growth = less energy output
2) nutrients are associated with surfaces (thus cells are on surfaces)!!
3) cooperation within community (easier in this env if one bacteria can make something required for another bacteria)
4) provides protection from antimicrobial agents, predators and the immune system
Where are biofilms formed?
1) in the environment
2) in industry
3) in medicine
*basically everywhere that has some sort of nutrient and moisture
Biofilms can grow on:
1) on medical equipments
2) on our organs!
3) in nature
Give distinct examples of each.
1) contact lens CASE and CATHETERS used for urine passage can be blocked by bacterial cells and matrix and lead to bad infections
2) S. aureus on CARDIAC tissue. P. aeruginosa in SPUTUM from a cystic fibrosis (CF) patient (example of biofilm not forming on real surface). - DENTAL PLAQUE (cause inflammation in gums –> heart problems)
3) Morning glory pool in yellowstone park (microbes colonize thermal pools and springs in the park & give them their distinctive and unusual colours).
1) When started studying biofilms at a molecular level in the late 1990s, what was the hypothesis they were testing?
2) What were the two approaches used to address this hypothesis?
Hypothesis: surface-attached cells (biofilm) express different genes compared to planktonic cells.
Approaches to address this hypothesis:
1) DNA microarray analysis
2) 2D gel protein analysis
A paper compared gene expression patterns of planktonic and biofilm cells using a P. aeruginosa DNA array.
1) How many genes did this array contain?
2) What did they find?
1) The array contained 5,500 of the predicted 5,570 genes.
2) 0.5% of the genes were activated in biofilms while 0.5% were repressed.
Activated genes included ones involved in protein translation and certain types of metabolism.
Repressed genes included ones involved in flagellar motility and secretion
Briefly describe how 2D gel electrophoresis occurs.
First, there is separation of proteins in first dimension (by charge).
This gel is applied to the top of the second gel.
Then, there is a separation in second dimension (by size).
A paper compared planktonic and biofilm cells expressed proteins using 2D gel analysis in P. putida.
What did they find?
P. putida biofilms major differences:
There are 15 up-regulated genes (pilis are up-regulated).
There are 30 down regulated genes (flagella is down-regulated).
Several DNA microarray and protein expression studies have been published.
What discoveries have these studies revealed thus far?
Planktonic cells and cells growing in biofilms do express different set of genes but there are NO GENES IDENTIFIED THAT ARE ONLY EXPRESSED IN BIOFILMS.
No biofilm markers have been identified!
This may be due to the use of different strains and growth conditions in these papers.
which statement is false?
1) most bacteria will form biofilms under certain conditions.
2) molecular aspects of biofilm formation tend to be studied for all bacteria.
2!
molecular aspects of biofilm formation tend to be studied for MEDICALLY or INDUSTRIALLY important bacteria.
Briefly answer the following questions of P. aeruginosa:
1) Describe the bacterial structure and strain.
2) What kind of pathogen is it?
3) What are the advantages of studying with this bacteria?
1) Gram negative, motile rod
2) Opportunistic pathogen - pathogen of immunocompromised and burn patients as well as people with cystic fibrosis (CF)
3) sequenced genome, genetically amenable, and best studied model for biofilms
Describe the steps of biofilm formation by P. aeruginosa
Planktonic cells initially attach to the surface in a reversible manner before transitioning into an irreversible attachment phase, leading to the FORMATION OF A MONOLAYER.
Then, there is the formation of a MICROCOLONY and then a MACROCOLONY (biofilm).
What kind of assay did O’Toole and Kolter use to determine which genes are important for biofilm formation?
What was their hypothesis?
O’Toole and Kolter used 96 well microtitre plate as assay system.
They used P. aeruginosa random transposon insertion mutant library and screened 2400 mutants. They identified mutants that did not form biofilms and determined which genes were affected in the mutants.
Hypothesis: specific genes control biofilm formation.
How did O’Toole and Kolter construct their random transposon-insertion mutant library?
They constructed their library by adding a transposon (tn) to wild type culture of P. aeruginosa.
Under specific conditions, transposon inserts RANDOMLY and only ONE TIME, into the chromosome of P. aeruginosa.
Among thousands of transposon-insertion mutants, should have an insertion in each non-essential gene (genes not required for survival).
If there is no biofilm, they can check which gene is mutated and know that it is important for biofilm.
Describe the 96-well Microtitre Plate Assay.
The 96-well microtitre plate assay involves adding cells to the wells, allowing them to adhere and form biofilms over a period of time (in this case, 10 hours).
After incubation, the wells are rinsed to remove non-adherent cells, and then a dye such as crystal violet is added to stain the remaining bacteria in the biofilm.
The absorbance at 600 nm (A600) is then measured at different time points to monitor the development of biofilms over time.
What mutants in O’Toole and Kolter paper did not form biofilms?
pilY1
pilB
flgK
What is the advantages and disadvantages associated with 96-well Microtitre Plate Assay?
Advantage: high throughput
Disadvantage: gross level (yes or no) - can not tell if little bacteria stuck on the walls
What is the flow cell system? How does it differ from microtitre plate assay?
Flow cell system is a flowing system where we can watch biofilm formation over time under the microscope.
There is a RESERVOIR with media that flows through the system. There is also a FLOW CELL with 4 channels/ grooves. Each channel is inoculated with different strains of bacteria at 37˚C. if bacteria is not attached, they will disappear with the flow.
A glass slip on top encloses channels; and this can be put on microscope and image bacteria.
It is less high throughput compared to the 96 well microtitre plate assay but it gives more information (less gross level).
Briefly describe what you would see under a microscope of wildtype biofilm formation in a flow cell over 7.5 hours.
Within 30 minutes, there is initial attachment.
In 1.5 hours, there is formation of a monolayer.
In 5 hours, cells start to come together forming microcolonies and in 7.5 hours, microcolonies become macrocolonies.
(T/F) flgK and pilB are important for biofilm formation at different stages.
True!
flgK mutants can’t attach.
pilB mutants can attach but are arrested at monolayer formation and can’t progress further.
What does flgK encode for?
flgK encodes a protein that is part of the FLAGELLUM.
Flagella is an extracellular structure important for swimming motility and/or ATTACHMENT TO SURFACES.
Thus flgK mutants can’t attach to form a biofilm!
What does pilB encode for?
pilB encodes a protein important for the synthesis of TYPE IV PILI.
Pili are short, thin proteinaceous fibers that cover entire surface of the cell. They are required for TWITCHING MOTILITY (used for testing where things are good to stick).
*PilB mutants can form a monolayer but can’t do twitching motility which is important for bacteria to come together thus can’t move forward to mature biofilm.
1) What kind of bacteria is E.coli?
2) What does it cause?
3) Do all strains of E.coli have the same virulence factors?
4) What is UPEC?
1) Gram negative bacteria
2) Causes diarrhea, dysentery, kidney failure, bladder infections and pneumonia
3) Virulence factors VARY with strain (one that causes diarrhea will not cause a UTI). Most strains are NON-pathogenic (live in our GI tract)
4) Uropathogenic E. coli (UPEC) causes UTI
(T/F) E. coli biofilm formation differs from P. aeruginosa.
False!
They follow the same steps: initial attachment -> monolayer formation -> microcolony formation -> macrocolony formation
In E. coli biofilm formation, what is required in the transition from
1) planktonic to initial attachment
2) initial attachment to monolayer formation
3) monolayer formation to microcolony formation
4) microcolony formation to macrocolony formation
1) flagella
2) Type I pili / Ag43
3) flagella
4) colanic acid
*colanic acid mutants form a microcolony but not macrocolony
What are the (3) properties of a mature biofilm?
1) Surrounded by EXOPOLYSACCHARIDE MATRIX.
2) HETEROGENEITY-BACTERIA throughout the biofilm experience different environments.
3) Resistant to antimicrobial agents as a whole.
1) What are the two main functions of the matrix?
2) How do we know this?
1) Structure and protection
2) Observation (microscopy) and identification of important elements of the matrix & then deletion of important genes to determine what their individual functions are
What is the matrix composed of?
1) polysaacharides (sugars)
2) DNA (eDNA secreted into matrix by lysed cells)
3) proteins (not much known about these)
