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PB > Lecture 4: Biofilms > Flashcards

Lecture 4: Biofilms Flashcards

1
Q

Whats a biofilm

A

Community of microbes attached to a surface (biotic or abiotic) encased in a matrix

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2
Q

Biofilms are usually…

A

Multispecies

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3
Q

Can most bacteria form a biofilm

A

Yes under certain conditions

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4
Q

What are most water bacteria like

A

Not free floating organisms but rather grow upon submerged surfaces

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5
Q

Biofilm developement cycle

A

Mature biofilm

Planktonic cells detach

Motility, adhesion and environmental signals

Acyl-HSL signal reatach to mature biofilm - autoinducer quorum sensoring

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6
Q

Why form a biofilm

A
  • 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
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7
Q

Can bacteria choose to be in biofilm, is it always more favourable to be in a biofilm

A

yes and no there are times when bacteria WANT to detach

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8
Q

Where are Biofilms found?
What tends to be studied more (in bold)

A

Environment

**Industry

Medecine**

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9
Q

WHats the dif between a planktonic cell and a biofilm cell
- Hypothesis
- Approaches to address this hypothesis

A

Surface attached cells (biofilm) express dif genes compared to free-swimming (planktonic) cells

DNA microarray analysis
2D gel protein analysis

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10
Q

What did DNA array analysis determine

A

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**

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11
Q

2D gel electrophoresis
- How to do it
- What it permited to determine (specificly what u have to remember from that slide)

Revise before exam

A

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)

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12
Q

After manys studies of DNA microarrays and protein expression of planktonic cells and cells in biofilm, what is the consensus?

A

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

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13
Q

What is an operunistic gram negative, rod bacteria that has a sequenced genome, is genetically amenable and best studied model for biofilms?

A

P.aeruginosa
aer ugi

nosa

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14
Q

What are features and advantages of pseudomonas aeruginosa

A

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

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15
Q

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)

A

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)

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16
Q

How were genes imporant for biofilm formation identified
- Who
- Procedure used

A

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

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17
Q

Random transposon-insertion mutant library how

A

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

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18
Q

What procedure was used to test formation of biofilm and how was it done
Revise before exam

A

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)

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19
Q

Review

Limitation of the 96 well procedure and what procedure is used instead (explain this other procedure)

A

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
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20
Q

Flagellum

A

flgK encodes a protein that is part of the flagellum (flagella is plural)

Important for swimming motility and/or attachement to surfaces

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21
Q

Pili

A

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)

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22
Q

What do flgK and pilB usually help with
Revise before exam

A

flgK helps reversible attachemetn to surface: flagella

pilB helps to stick to other cells to form microcolonies: pili 4

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23
Q

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

A

E.coli

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23
Q

Descibe E.coli

A
  • 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
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23
Q

Names of strcutures that help with E.coli biofilm formation
Revise before exam

A

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

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23
Q

Properties of mature biofilm?

A
  • Surrounded by exopolysaccharide matrix
  • Heterogeneity-bacteria throughoout the biofilm experience different environments
  • Resistant to antimicrobial agents
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23
Q

Function of matrix and how do we know this

A
  • 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

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24
Q

Matrix composition

A

Poysaccharides
DNA (eDNA for extracellular DNA)
Proteins

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25
Q

Polysaccharides in the P.aerurginosa
Revise before exam

A
  • Alginate
    +overproduction occurs in CF patients
  • PsI
    +Mannose and galactose rich polysaccharides
  • pel
    +Involved in pellicle formation (floating biofilm)
    +Glucose rich polysaccaride
26
Q

eDNA
- Where does it come from
- Whats its function

A

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)

27
Q

Heterpgeneity
- What causes it
- Why does it matte

A

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

28
Q

Planktonic antibiotic resistance resistance mechanisms

A

Intrinsic (part of genome)
- Low outer membrane permeability
- Action of efflux pumps
Acquired (through mutation)
- Upregulation of drug efflux pumps
- Alternation of antibiotic target

29
Q

Biofilm-specific resistance mechanisms

A
  • Multiple mechanisms
  • Not based on mutations
  • Matrix (eDNA)
  • Biofilm-specific gene expression
30
Q

Beneficial Biofilms

A

In:
- Water treatment
- Energy production (microbial fuel cells)
- Nitrogen fication
- Biocontrol (protecing crops)

31
Q

Wastewater treatment

A

Primary, secondary and tertiary

In the aeration tanks, biofilm and planktonic bacteria break down organic matter (secondary)

32
Q

Microbial Fuel Cells (MFCs)

A

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

33
Q

Plants and biofilms

A

Alfama and soybean fix nitrogen

p.fluorescens protect tomatoes from rot

34
Q

Nitrogen fixation

A
  • 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
35
Q

Examples of plants and bacteria positive ineractions

A

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

36
Q

Problematoc biofilms exmaples

A

In industry:
- Clogging and corroding pipes
- Marine biofouling (on ships)
In environment:
- plant pathogens
In medecine
-65% of human bacterial infections are biofilm based

37
Q

Biofilms in Medecin

A
  • 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)
38
Q

Examples of diseases caused by biofilms in medecine

A

Recurrent urinary tract infections by E.coli

CF by p. aeruginosa

39
Q

A model of biofilm development

A

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

40
Q

When are bacteria harmful throughout biofilm stages

and why are biofilm cells recurring natured

A

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

41
Q

Chronic vs acute infections

A

actue: plaktonic cells expressing virulence factors

Chronic (long lasting): Don not express virulence factors

42
Q

Whats focused on when studying biofilms and diseases

A

Providing evidence that the infection is BIOFILM based the to identify approaches to eliminate the biofilm

43
Q

What is CF
Revise before exam

A
  • 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)
44
Q

What evidence is there for biofilm in the CF lung

A

Based in quorum-sensing autoinducer profiles (indicative of biofilm growth as opposed to planktinik growth)

Microscopy

45
Q

EXPLAIN

Quorum sensing: A bacterial cell density sensing system

A
  • 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
46
Q

Relationship autoincucer and p.aeruginosa

A

in this bacteria there are 2 dif autoinducer types and THEIR RATIO DEFFER depending on whether they are growing on:
- Planktonik cells
or
-Biofilms

47
Q

How does p.aeruginosa biofilm in CF lung persist

A

Virulence factor type III secretion system that injects proteins into host cells is not expressed when in biofilm

48
Q

How does p.aeruginosa cause damage in CF lung?

A

INDIRECT damage done by biofilm:
- Immune cells are recruited but cant kill biofilm infection
- Protease, free radicals produced by immune system damages lung tissue

49
Q

What is the CF lung environment and how is this promising

A

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

50
Q

p.aeruginosa biofilms aerobic and anaerobic conditions how it is studied

A

Anaerbobic: biofilm growth otherwise no

2D gel analysis spot 29 is present in aerobic biofilm but not in aerobic and this is OprF (porin)

51
Q

oprF importance for biofilm formation in P.aeruginosa

A

Very little biofilm formation when it is not present

52
Q

How do mutations accumulate over time of P.aeruginosa in CF lung

A

Slow growth = stressful conditions = accumulation of mutations in the bacteria

53
Q

What mutations accumulate over time in
as P.aeruginosa adapts in CF lung
Revise before exam

A

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)

54
Q

UTI’s

A
  • Considered to be 1 of the most COMMON bacterial infections
  • Tends to recur despite antibiotic therapy
  • Most common isolate is uropathogenic E.coli (UPEC)
55
Q

Experiment used to proove that UTI’s are biofilm based infections

A

Used mice. DId z-series to show bacteria are located thoughout the pod and biofilm is formed

56
Q

Biofilm formation Ecoli, important genes and role

A

Type I pili and Ag43 are impotant for E.coli to attach to surface

57
Q

control of biofilm formation

A

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

58
Q

2 major QS systems in p.aeruginosa
Revise before exam

A

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

59
Q

Is QS involved in biofilm formation?

A

a lasI mutation affects biofilm formation:
- WT: macrocolony
- lasI mutant: no macrocolony
- LasI mutatn + autoinducer: macrocolony

60
Q

QS inhibitor
Revise before exam

A

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

61
Q

What are some QS controlled processes

A

Biofilm formation

Fruiting body formation

Production of antibiotics

Expression of virulence factors

Expression of bioluminescence

Motility

62
Q

What techinc was used to figure out how the seaweed QSI worked, like what process it shut dows

A

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

63
Q

How are biofilms studied

A

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

64
Q

How can you detect a biofilm

A

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

65
Q

Biofilm detachement

A

Natural part of biofilm lifecycle and get be released in clumps or single cells

66
Q

Identification of a detachement factor

A

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

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