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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
Salmonella alfalfa sprouts attachment
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
26
Salmonella attachment mechanisms
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
Salmonella curli amyloid formation
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
E. Coli on spinach leaves
Attach to stoma (guard cell) Attach to other parts too
29
GFP conclusion
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
Microscopic studies conclusions
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
Why do they attach strongly and resist disinfection?
Flagella - motility and attachment Other organelles - Fimbriae, pili? Altered physiology - qs, RpoS
32
Expediency vs relevance, artefacts?
High glucose - boosts many biofilms - in vivo glucose low unless diabetic High nutrients - in vivo (
33
Monitoring and modelling
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
Monitoring and modelling examples
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
What parameters
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
Typical of monocultures in rich media
Mushroom like Stalk and cap Water channels
37
Open architecture structure
Heterogeneous - differences in colours Stack or fronds of microcolonies (waves around with water flow, viscar elastic) Haven against environment and predators
38
Microtiter plate assays for biofilm formation
Culture medium Inoculate See if they attach Supranatants Stain - colour intensity (more glucose more biofilm) Extract and spectrophotometer
39
High throughput screening for antimicrobials or mutants
96 well plate screening See if coatings reduce attachment
40
MBEC high throughput (HTP) assay
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
Lab flow cells
Not robust Low shear Slow Flow Linear gradient effects? Ok for direct microscopy but need to be transparent
42
Removable flow cells
Robust Higher shear Detach and look at biofilms
43
Transparent substrata excellent for microscopy
Differentiation and death in microcolony Cell death in centre via flow cells
44
ATR-FTIR ge Crystal flow cell
Infrared Scans of wavelength and so spectrum Render into chemical bonds that correspond eg amide bonds, fatty acid etc
45
Ribbons device
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
On line device
Hedgehog biofilm monitoring device
47
KIWA biofilm monitor (glass)
Glass slats could be removed
48
Sentinel coupons
Metal or plastic Unscrew and so have inserts Replace and restart flow and investigate biofilm
49
Annular reactor models
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
Annular reactor
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
Chemostat models
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
Microbial film formation: dental plaque
No sucrose or glucose needed Mouthwash didn’t effect biofilms as much
53
Chemostat models for water studies
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
Enhanced EPS production on copper
Usually microbes don’t survive on copper but those that survived had greater EPS
55
Safe haven for pathogens
Indicator bacteria - E. coli Pathogens - Ecoli Opportunistic pathogens - ps. Aeruginosa Microaerophiles- h pylori Protozoa - c. Parvum
56
Transition analysis
Analyse selection of mutant organisms as cultures adjust from one steady state to another
57
CDC reactor
Plastic coupon holder Used in labs Surface area to volume ration
58
Propella reactant
Stunts Control flow rate Linear flow
59
Propella attributes
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
Drop flow biofilm fermenter
Dental plaque Supports biofilms on slides that are maintained with growth medium drip wise
61
Constant depth film fermenter
Static arm so “tongue” So inside plugs, constant depth Biofilm formation in plugs It rotates
62
Perfumed biofilm reactor
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
Sorbarod biofilm fermenter
Low cost materials Syringe for medium feed
64
Conclusion of models
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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