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Microbio & Biofilms > Biofilms Of The Phylloplane And Generation Menthods > Flashcards

Biofilms Of The Phylloplane And Generation Menthods Flashcards

1
Q

Rhizosphere

A

Below ground habitat colonised by microbes

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2
Q

Phyllosphere

A

Aerial plant habitat colonised by microbes

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3
Q

Phylloplane

A

Microbes live on the phylloplane leaf surface
Inhabitants are called epiphytes

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4
Q

Phyllosphere global ecosystem

A

Terrestrial leaf surface area that might be colonised by microbes - approx 640 million sq km
I’m aggregate these bacteria are sufficiently numerous to contribute to many global processes of importance as well as behaviour individual plants

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5
Q

Plant microbe interactions

A

Most work involves rhizosphere
Nodules and nitrogen fixation (flavonoids/nod genes)
Agrobacterium (ca2+ dependent adhesion, att genes, EPS, cellulose fibres)
Attachment of e.coli and salmonella go roots (curli)
Internalisation of salmonella through lesions

Work on Phyllosphere
Plant pathogens
Zoonotic pathogens

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6
Q

Phylloplane global ecosystem

A

Fluctuating environmental stresses (uv, temp, desiccation), host to diverse microbial colonists and plant pathogens
Work mainly considered economic and sociological impacts of plan pathogens and spoilage microorganisms
Disease outbreaks can be undetectable
Limited and controversial due to limitations in analytical techniques

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7
Q

Leaf structure

A

Waxy cuticle
Stoma for gas and water exchange
At top and bottom of leaf
So colonies on both top and bottom of leaves

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8
Q

Citrus canker

A

Evidence for subsurface invasion via stomata

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9
Q

Complex bacterial communities on leaves of perennial rye (lolium perenne)

A

Pseudomonas flourescens 20.12%
Xanthomaonas campestris 19.64%
Listeria spp. 4.02%

Food company always issues with listeria

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10
Q

Complex bacterial communities on leaves of olive (olea Europea)

A

Pseudomonas syringae 51%
Xanthomonas campestris 6.7%

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11
Q

Human guy pathogens found on fresh produce

A

E.coli - apple juice, bean sprouts, cabbage,celery, coriander, cress, lettuce
Salmonella - aubergine, bean sprouts, celery, cabbage, lettuce, orange juice, spinach
Campylobacter - lettuce, mushrooms, potatoes, parsley, spinach
Listeria monocytogenes- bean sprouts, cabbage, cucumber, lettuce
Shigella - celery, lettuce, melon, parsley

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12
Q

Pathogen vehicles onto the leaf surface

A

Soil - manured
Slugs & nematodes fecal deposits, pigeon fecal
Pigs walked through the salad fields deficating
Irrigation of water

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13
Q

Is it difficult to clean and package leaves?

A

Food companies can’t keep out frogs/toads, bugs etc

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14
Q

Methods of testing

A

Ex situ - sample preparation and culture recovery
Presence of specific bacterial species determined by playing onto selective culture media and incubation
Backbone of many published studies and mana diet use by QC labs to meet international regulations

But
Non culturally species/strains
Sun lethally stressed cells may not grow within incubation period
Accuracy dependent on efficient recovery from sample being tested (sample prep very important)

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15
Q

I’m situ detection

A

EDIC/EF
Patented advanced microscopy

Phylloplane: convoluted complex environment

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16
Q

Unwashed spinach leaf

A

EDIC
Material: Bacteria and microcolonies or is it soil?
DAPI - so DNA material

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17
Q

Washed leaves

A

Chlorine water - not a lot of water so recirculate so control water quality
Oxidant so close guard cells so microbes stuck inside cell
Biofilms still found and viable

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18
Q

Tracking human pathogens on leaves: GFP bacteria (salmonella)

A

Immediately after inculcation - many bacteria in cell margins but also well spread over leaf surface
Within 2 hrs - bacteria predominantly present at cell margins and within stomatal apertures

Active migration - what substance is attracting the bacteria? Highly motile, can swim inside, makes decision

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19
Q

Subsurface migration

A

Coriander
On or below surface of vein

First outside of leaf
Couple days inside of leaf

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20
Q

Potential attachment mechanisms to the Phylloplane

A

Flagellum
Pili
Fimbriae

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21
Q

Salmonella attachment mutants

A

deleted crl and deleted csgB (curli specific gene) mutant strains partly lacking curli Fimbriae
Deleted rpoS, crl and csgB mutant strains lacking curli finbrae
flhC mutant strains lacking flagella

Abiotic surface - put salmonella in and see if attached
Crl mutant still stack
RpoS, csgB and double knockout not so well

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22
Q

Salmonella curli Fimbriae mutants show little difference in attachment to spinach Phylloplane

A

10^6
So not as important in Phylloplane so flagellum?

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23
Q

Attachment of salmonella typhumuriun flhC mutant to abiotic surface

A

Mutant less
Temperature has impact as well (30 has greatest attachment) 

Leaf surface - 37 temp great difference

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24
Q

Salmonella attachment mechanisms

A

Deleted rpoS, crl and csgB mutant strains lacking curli Fimbriae or flhC mutant strains lacking flagella are unable to attach to abiotic polystyrene

Only flhC mutant showed reduced attachment in Phylloplane but impacted by temp

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25
Q

Salmonella alfalfa sprouts attachment

A

2/3 genetic insertions in genes not seen before as well as:
CsgB (intergenic region)
CsgD (transcriptional regulation of LuxR superfamily - QS)
RpoS (stationary phase sigma factor)

CsgD and RpoS regulate curli and cellulose production
Not that much of a drop in attachment tho
CsgB can play important role in attachment of S. enterica to plant tissue - thing aggregative fimbriae

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26
Q

Salmonella attachment mechanisms

A

CsgD regulates the production of curli
Also positively regulates AdrA which up regulates bcs genes responsible for cellulose production
Also regulates bapA that produce BapA protein (biofilm associated protein in presence of ca2+ helps attach)

27
Q

Salmonella curli amyloid formation

A

CsgA is subunit is an amyloid protein
Stack up and make the curli

CsgD has regulation effect through phosphorylation of apartic at 59 position
Porin subunits come through and transporter system to make curli

RpoS unregulated crL genes normally which up regulates adrA which makes cellulose
Knock out RpoS lose positivite regulatory effect and no longer make cellulose and curli

28
Q

E. Coli on spinach leaves

A

Attach to stoma (guard cell)
Attach to other parts too

29
Q

GFP conclusion

A

When motile salmonella land on lead surface, free water on surface allows them to seek refuge in cell margins and stomata
Subpops of cells may have different colonisation strategies
Refuges less hostile in terms of water availability and protection from solar irradiation, sanitisers and washing/detachment method
Clear evidence for biofilm/EPS on leaves
Salmonella co-localisation

30
Q

Microscopic studies conclusions

A

Salad leaves naturally host to many bacteria which aggregate on leaf surface and visible by directing light microscopy
Tightly attach and resist removal by waylaying and stomaching
Form biofilms, complex 3D structures which help them adhere better and shield from effects of chemical sanitisers eg chlorine

31
Q

Why do they attach strongly and resist disinfection?

A

Flagella - motility and attachment
Other organelles - Fimbriae, pili?
Altered physiology - qs, RpoS

32
Q

Expediency vs relevance, artefacts?

A

High glucose - boosts many biofilms - in vivo glucose low unless diabetic
High nutrients - in vivo (<Fe), environment (<C, N,P)
O2, neutral pH in vitro or in vivo relevance as in real life anerobic

33
Q

Monitoring and modelling

A

Monitor biofilm development in relation to physicians chemistry of
Pipe material, roughness, age, corrosion
Temp
Disinfection residual
Influence of perturbations eg nutrient ingress following cracked pipe etc
Track of pathogens

Access quality (bio stability) of water and relation to biofilm potential on line or off line systems

Sites
Mains distribution supply - low temp 4-20
Building plumbing systems 20-60

34
Q

Monitoring and modelling examples

A

On line - in situ
Direct eg Robbies type devices in mains pipe, side loop pipes, end of tap biofilm sensors

Off line - pilot scale
Several km rigs eg Nancy, Kemptom

Off like - lab models
Several km rigs eg Nancy,
Flow cells, chemostats, annular reactors l, propella

Problems of scale and artefacts
Can small flow cell provide meaningful data relevant to river or large mains
Investigate define parameters

35
Q

What parameters

A

Chemical analyses: largely recovered biofilm

Microbiological:
Intact biofilm- microscopy eg EDIC, DAPI, CTC, Bac light
Recovered/homogenised biofilm - microscopy, cell cytometry, community analysis eg DGHE, culture

36
Q

Typical of monocultures in rich media

A

Mushroom like
Stalk and cap
Water channels

37
Q

Open architecture structure

A

Heterogeneous - differences in colours
Stack or fronds of microcolonies (waves around with water flow, viscar elastic)
Haven against environment and predators

38
Q

Microtiter plate assays for biofilm formation

A

Culture medium
Inoculate
See if they attach
Supranatants
Stain - colour intensity (more glucose more biofilm)
Extract and spectrophotometer

39
Q

High throughput screening for antimicrobials or mutants

A

96 well plate screening
See if coatings reduce attachment

40
Q

MBEC high throughput (HTP) assay

A

Static well so gravity so sediment down
Peg wells so 3D
Adherence and shedding of biofilm to pegs so not gravity
But still static system

41
Q

Lab flow cells

A

Not robust
Low shear
Slow Flow
Linear gradient effects?
Ok for direct microscopy but need to be transparent

42
Q

Removable flow cells

A

Robust
Higher shear
Detach and look at biofilms

43
Q

Transparent substrata excellent for microscopy

A

Differentiation and death in microcolony
Cell death in centre via flow cells

44
Q

ATR-FTIR ge Crystal flow cell

A

Infrared
Scans of wavelength and so spectrum
Render into chemical bonds that correspond eg amide bonds, fatty acid etc

45
Q

Ribbons device

A

In line
Robust
High pressure
Assess different materials
Crystal

Modified ribbons device
Lab model
Not robust, low pressure
Reproducible
Shear effects at start linear gradient?
(Data taken from middle studs)

46
Q

On line device

A

Hedgehog biofilm monitoring device

47
Q

KIWA biofilm monitor (glass)

A

Glass slats could be removed

48
Q

Sentinel coupons

A

Metal or plastic
Unscrew and so have inserts
Replace and restart flow and investigate biofilm

49
Q

Annular reactor models

A

Defined environment - physico chemistry
pH, eh, nutrients
Reproducible but not as well mixed as chemostat
Open flow system - model pipes, tanks
Safe, no leaks (suitable for dangerous pathogens)
Suitable for biofilm structure and function, pathogen survival, biocide efficiency, material biofouling

But eddy current effects: x and y gradients

Glass reactor with rotating inner drum, glass slides inserted, restart motor and flow
Shear can be controlled
Change in fluent water quality

50
Q

Annular reactor

A

flow 4mL/min
Exachange if the media: 9.3 x per day
Rotation of inner cylinder: 200rpm
Flow velocity: 0.45m/s
Shear force: 2226 Re

Biofilm gradient - start of surface compared to other due to forces shearing

51
Q

Chemostat models

A

Defined environment - physico chemistry
pH, eh, nutrients
Reproducible but not as well mixed as chemostat
Open flow system - model pipes, tanks
Safe, no leaks (suitable for dangerous pathogens)
Suitable for biofilm structure and function, pathogen survival, biocide efficiency, material biofouling

Data obtained as found in field
Possible issues - shear forces not completely controlled but no eddy current affect on biofilms

52
Q

Microbial film formation: dental plaque

A

No sucrose or glucose needed
Mouthwash didn’t effect biofilms as much

53
Q

Chemostat models for water studies

A

Flow through design with tap water as feed
Ability to change in fluent water quality by adding different feed solutions (nutrients, disinfectants)
Capability to control shear stress by changing rotational speed of stirrer
Ability to control hydraulic retention time through in fluent flow rate
So shear stress and hydraulic retention time set independently
Biofilm sampling by removal of coupons form reactor

54
Q

Enhanced EPS production on copper

A

Usually microbes don’t survive on copper but those that survived had greater EPS

55
Q

Safe haven for pathogens

A

Indicator bacteria - E. coli
Pathogens - Ecoli
Opportunistic pathogens - ps. Aeruginosa
Microaerophiles- h pylori
Protozoa - c. Parvum

56
Q

Transition analysis

A

Analyse selection of mutant organisms as cultures adjust from one steady state to another

57
Q

CDC reactor

A

Plastic coupon holder
Used in labs
Surface area to volume ration

58
Q

Propella reactant

A

Stunts
Control flow rate
Linear flow

59
Q

Propella attributes

A

Combines best of chemostat l, annular reactor
Defined, reproducible
Good mixing
Constant shear, no apparent artefacts or gradients
Shear stress and hydraulic retention time set independently
Robust connect to mains supply or lab model

Problem
Leaks so bad for pathogen studies

60
Q

Drop flow biofilm fermenter

A

Dental plaque
Supports biofilms on slides that are maintained with growth medium drip wise

61
Q

Constant depth film fermenter

A

Static arm so “tongue”
So inside plugs, constant depth
Biofilm formation in plugs
It rotates

62
Q

Perfumed biofilm reactor

A

Control growth rate in biofilm
Collect baby cells in eluate
Good for studying u and antimicrobial resistance but force medium through biofilm affecting structure

63
Q

Sorbarod biofilm fermenter

A

Low cost materials
Syringe for medium feed

64
Q

Conclusion of models

A

Favour own models
Most suffer issues
Some only appropriate for lab
Some for real world
Beware of artefacts

Microbio & Biofilms (8 decks)

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