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Microbio & Biofilms > Mutation And Diversification In Biofilms > Flashcards

Mutation And Diversification In Biofilms Flashcards

1
Q

Biofilms are highly heterogenous structures

A

Eg Stalk and cap
Microcolonies in cystic fibrosis the same not just in the lab
Aggregates and microcolonies structure

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2
Q

Antimicrobial tolerance in bacterial biofilms

A

Matrix impendence eg gent
Enzymes eg beta lactemase
Biochemical changes eg periplasmic glucans
Metabolic gradients eg ciprofloxacin and tobramycin only fill cells in metabolically active top layer
Physiological sun pops eg persisters

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3
Q

What causes a subset of cells within the biofilm pop to develop into microcolonies?

A

Challenges of biofilm antirestance and tolerance

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4
Q

Understanding biofilm development

A

Identification of gene systems / mechanisms involved
Possible to manipulate physiological status

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5
Q

Biofilm flow cell

A

Medium
Pump
Flow cell
Waste

“Continuous culture” and can image and watch biofilm grow, nutrient medium flowing, bacteria can washed through so only watching behaviour of biofilm and not planktonic

Medium, bacteria, surface seeking and attach to cover slip, biofilm formation

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6
Q

Biofilm structure development

A

2 Fluorescent protein reporters
Same organism but 2 pops one blue one yellow
Mixed population
Separated itself
Why? Global structure from single cell and grown into colony

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7
Q

Phenotypic variation in biofilm derived bacteria

A

Bacteria develop phenotypic differences from each colony
Eg morphology, small phenotypes, wrinkled, different pigmentation, motility
Doesn’t happen in planktonic cells so biofilm specific phenomena

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8
Q

Phenotype arrays show different substrate - utilisation profiles of CF p.aeruginosa isolates

A

Purple - uses carbon source
Wt and SC 1 biofilm derived, lost ability to use some carbon sources and gained use of others
Metabolic and phenotypic change

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9
Q

Is there a role for mutation and genetic variation in biofilm structure?

A

Maybe
Mutations
Driving phenotypic variations and structures of biofilms

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10
Q

Is there a role for mutation and genetic variation in microcolony growth

A

P. Aeruginosa DNA mismatch repair deficient strains
-WT
-MutS
-mutL

Mut strains frequency 100x greater than WT
P. Aeruginosa mutator strain exhibit enhanced microcolony development
Biofilm bio volume increased in mutator strains
Increased size of microcolonies

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11
Q

Microcolony initiation

A

PAO1 DNA repair mutants

Mutation frequency increases and increases microcolony formation (hetergenous micro colonies)

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12
Q

Deep sequencing of p. Aeruginosa biofilm pop - observation of genome evolution

A

Genomic sequencing of pop in biofilm
Which genes changed by biofilm
Grow biofilm, extract DNA, sequencing, compare to WT strain, mutation discovery

115 SNPs
Encodes bacteriophage region, lots of mutations (not key)
Lots of mutations across whole genome
Single nucleotide polymorphisms I’m coding and no coding regions

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13
Q

Which genes affected by nonsyn mutations

A

PA4341
pA263

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14
Q

Mutation detection in situ

A

GFP +1 reversion system pMDGFP
Green fluorescent protein gene, frame shift mutation, can’t express functional so can’t flow, place into p. Aeruginosa biofilm
Mutation and glows green again if mutation happened
Some did this

Increased 100-1000 fold higher in microcolony cells than non

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15
Q

Darwinian processes in biofilm development

A

Microcolonies foci for genetic mutation and evolution
Microcolony growth may involve mutation selection

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16
Q

Model for microcolony develop

A

Primary mutation
Clinal expansion
Secondary mutation

Clinal selection

17
Q

Analgy: clinal succession and solid tumour development

A

Evolution of cancer as multiple mutations
Proliferation of beneficial (selective)
Lots of commonality of the selective ones even tho random

18
Q

Active dispersal in biofilms

A

Some cells can do this
Intrinsic mechanisms - bacteria escape and colonise new sites, hollowing out of colonies
Gaps and pores and so escape

Happens in multi species dental biofilms & Waste water treatment granules

19
Q

Understanding genetics and physiology of biofilm dispersal

A

Identify gene systems involved in dispersal for targeting
Possibility to manipulate physiological status of biofilm to enhance/inhibit dispersal

20
Q

Pseudoalteromonas tunicata (D2)

A

Obligate Marine bacteria
Colonised and biofilms on marine plants eg ulva lactuca
Produces bio active compounds that inhibits marine fouling organisms eg algal spores
Forms microcolony based biofilms in lab and vivo

21
Q

Pseudoalteromonas tunicata biofilm

A

Biofilm lysis, detachment and dispersal
Inside colonies die
Biofilm parts detach

22
Q

P. Tunicata auto lytic protein AlpP

A

Detected in waste effluent of biofilms ~ 3 days old
Autolytic, produced in biofilm

23
Q

AlpP mediates cell death in P. Tunicata biofilms

A

Knock out gene
Don’t see lysis and death
Remains viable

24
Q

AlpP mode of action

A

Production of hydrogen peroxide from oxidation of L-lysine
Lysine + 02+ H20 = 6 amino 2 oxo hexanoate + NH3 + H2o2

Amplex red reagent react with H2O2 to produce red fluorescent oxidation product resuforin

Hydrogen peroxide detected in p. Tunicata biofilms

25
Q

AlpP is required for biofilm dispersal in p. Tunicata

A

Facilitates detachment of subset of survival and dispersal of some cells
Kills others during that break up

Mutant - biofilm just sits there, no dispersal

26
Q

AlpP occurs in several gram negative organisms

A

Marinomonas meditteranea

AlpP and it’s homologues produce hydrogen peroxide and induce lysis, dispersal and phenotypic variation

27
Q

Paudononas aeruginosa

A

Live dead stain
Similar lysis of subset of cells as biofilm develops like P. Tunicata

28
Q

Microcolony differentiation linked to production of reactive oxygen species

A

Dihydromine- ROS detective

Production of peroxynitrite (ONOO) in microcolonies 
Supraoxide and nitric oxide

29
Q

NirS expression in mature p. Aeruginosa biofilms

A

Reporter on NirS via transcriptional fusion
Bright fluorescent
So expressed in some colonies

Involvement of nitric oxide in cell death and dispersal events in p. Aeruginosa
Add SNP (nitric oxide) (low doses) causes dispersal when added to the biofilm. Makes rest of biofilm more sensitive to antimicrobials

30
Q

Nitric oxide induced dispersal of p. Aeruginosa biofilms

A

SNP added at low dose diapers
Too much then reverse effect and even more biofilm formation (protect themselves?)

31
Q

Microarracy analyses: p. Aeruginosa responds to low lvls NO

A

Increased motility genes eg flgG
Less adhesion and biofilm relevant genes eg cupP, C, EPS Pel genes, alg genes
Genes containing GGDEF or EAL domains - cyclic-di-GMP turnover? Eg PA1181, bdlA

32
Q

Cyclic- di- GMP regulates biofilm vs planktonic lifestyle across broads range of bacteria

A

Increase cyclic-di-GMP = biofilm formation
Reduce cyclic-di-GMP = planktonic

INTRACELLULAR

33
Q

Cyclic-di-GMP turnover

A

Conserved domains
GGDEF - guanylate Cyclades
EAL - phosphodiesterase (breaks down to GMP)

PAS or Nhox- bind and respond to NO in both eukaryote and promaryites

34
Q

Influence NO on c-di-GMP

A

Reduced c-di-GMP
Causes dispersal in p. Aeruginosa
Turns off biofilm traits

35
Q

Cystic fibrosis

A

Lethal hereditary disease
Autosomal recessive mutations in CF transmembrane conductance regulator (CFTR)
Increase mucous production so bacterial colonisation, poor ciliary clearance
Chronic P.aeruginosa key factor in death

36
Q

Adjunctive NO in cystic fibrosis - hypothesis

A

Low dose NO to lung
Reduc pseudomonas aeruginosa by reducing antibiotic tolerance of biofilms
Enhance efficacy of antibiotics
Reduce ineffective treatment and burden
Improve respiratory function
Improve quality of life

37
Q

Dispersal of p. Aeruginosa clinical isolates in CF- sputum

A

NO = break up colonies

38
Q

Phase 2 pilot study - reducing antibiotic tolerance with nitric oxide (RATNO)

A

Placebo and NO
Patients didn’t know which
Reductions with NO adjunctive so proof of concept
Seen through FISH (targeted probe to p. Aeruginosa biofilm fluorescing green)
Can’t remove completely but proof of concept so promising

Microbio & Biofilms (8 decks)

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