Biofilms 1 Flashcards
Infectious Diseases fall into what two categories?
Function of susceeptibility of he host:
- immune competent/compromised
- immunisations
- age
-trauma
- genetics
- antimicrobial therapy
Relates to the mechanism of bacterial pathogenesis
- secretion of factors (toxins)
- direct host cell manipulation
- evasion of host immune response
What are our different types of virulence factors
Adherence (adhesins) and colonisation factors
Invasion factors
Capsules
Siderophores
Endotoxins
Exotoxins
What is the main virulence factor bacteria has evolved for survival and adaptation?
Biofilm formation
Bacteria that form biofilm exist in two forms what are these?
Planktonic cells
Biofilm
How do planktonic cells differ from biofilm
Planktonic cells are sensitive while biofilm is not
What are planktonic cells
Single cell/bacteria or individual cells independently existing
Think of how bacteria grow in the lab
If planktonic cells are single cells what is biofilm?
In a biofilm cells are attache to each other or to a tissue or device -> they create a community
This attachment is irreversible
Why is biofilm more resistant than planktonic cells
Biofilms are sessile
Theyre sticky - gloopy matrix that coats the biofilm
Theyre stubborn – difficult to remove from a device or from the healthcare environment
Theyre hard to treat with antibiotics
Theyre hard to remove with disinfectants
What are the three S’s of biofilm
Sticky
Stubborn
subversive
What is a biofilm
Communities of bacteria attached to a surface
Define the two different lifestyles of biofilm
Planktonic (free-swimming), nomadic bacteria
Surface attached bacteria that form sessile communities called biofilm
Define biofilm, what three things are needed in order to called a bacteria a biofilm
Microbially derived sessile community characterised by cells that are irreversibly attached to a substratum or interface or to each other
And are embedded in a matrix of extracellular polymeric substances (EPS) that they have produced
And exhibit an altered cell phenotype with respect to growth rate and gene transcription
What does EPS stand for?
Extracellular polymeric substances
What is the role of EPS in biofilm structure
Biofilms self produce a matrix of hydrated extracellular polymeric substances that form their immediate environement
They provide mechanical stability to the bacteria,
Help mediate bacterial adhesion
The 3D polymer netweork interconnects and immobilised biofilm cells
Role in tolerance to antimicrobials-defence barrier
Development and dispersion
What is the EPS made of?
Polysacchrarides - alginate
Proteins
Nucleic acids
Lipids
What specifics form the EPS in the biofilm of P. aeruginosa?
Polysaccharides such as :
- alginate
- Psl for adherence to EC
- Pel -> SCD
Nucleic acids such as eDNA
What are the three functions of eDNA nucleic acids that form bofilm in P. auriginosa
Chealate cations
Disrupts bacterial membranes
DNAse dispersal of early biofilms
What does the alginate of P. aeruginosa biofilm do?
Its a polysaccharide thats associated with chronic stages of infection
What does the Psl and Pel of P. aeruginosa biofilm do?
These are important in the initial stages of biofilm production
What genes are responsible for alginate, Psl and Pel in P. auriginosa biofilm
pslA
alg8
pel
What did deletion of pslA and alg8 result in, in P. auriginosa biofilm?
this resulted in cells that overproduced Pel
What did deletion of pslA OR alg8 result in, in P. auriginosa biofilm?
This resulted in loss of characteristic mushroom structure in later biofilms
What did deletion of pslA and pel result in, in P. auriginosa biofilm?
Bacteria lost their ability to form biofilms altogether
What are the four stages of biofilm formation
Monolayer formation:
1. reversible attachement
2. ireversible attachment
- Microcolony Formation
- Macrocolony Formation
- Mature biofilm formed
NB: United we stand, divided we fall
What is responsible for controlling the motility-sessility switch in P. auriginosa?
Cyclic dimeric guanosine monophosphate
How does cyclic dimeric guanosine monophosphate (c-di GMP) control motility-sessility switch in P. aeruginosa?
High c-di GMP initiates biofilm formation
Low c-di GMP brings about dispersion
What is c-di-GMP?
Cyclic dimeric guanosine monophosphate
A small nucleotide-based signalling moelcule in bacteria that functions as a second messenger mediating a wide range of bacterial processes such as cell motilitty and biofilm formation
What is step 1 of biofilm development?
Conditiong:
- over time protein and polysaccharides deposit on surfaces
- other factors such as pH, atmosphere, temperature, oxidation and sheer forces are involved
What is step 2 of biofilm development
Reversible attachement:
- presesnce of flagella is important here
- type III pili twitching, brings bacteria in close contact
What is stage 3 of biofilm development?
Irreversible attachment:
- Cells in close contact to one another and the substrate
- Loss of motility
- Activation of quorum sensing system
What is quarum sensing
The regulation of gene expression in response to fluctuations in cell-population density
It controls the expression of numerous virulence factors including exotoxin A, elastase, proteases, haemolysin and siderophores
Why is quorum sensing needed for biofilm production?
As we need downregulation of some of the other virulence factors such as loss of motility
What is stage 4 in biofilm development
Maturation I:
- cells become layered
- film thickens to 10um
What is stage 5 of biofilm development?
Maturation II:
- Cell clusters averagve thickness 100um -> can take up to 6 days for Pse to devlop this
- Majority not attached to surface
What is stage 6 in biofilm development?
Some cells alter, regain motility and leave from the centre of the film
Absence of dense extracellular material at center
NB: dispersal v.important in healthcare settings
How do the bacteria in a biofilm below depth of 30u differ from those above?
The microcolony below this point is anaerobic utilising nitrate as a terminal electron receptor
List the 6 stages in biofilm development
Conditioning
Reversible attachment
Irreversible attachment
Maturation I
Maturation II
Dispersal
Desribe the structure of a mature biofilm
A channel flows just underneath the biofilm, between surface and biofilm -> biofilm extends collumns down through this
Main body of biofilm is composed of cell clusters with a void in the centre
Tail end of biofilm consists of streamers of clusters
Bulk fluid surrounds the biofilm
MB: should be able to draw this
Where might you find P. auriginosa biofilm embedded in host-material bs surface attached
Host material embedded as in cystic fibrosis wounds
Surface attached as in implants, catheters etc
What are the four reasons bacteria will form a biofilm?
Defense
Favourable habitat
Community
Default mode
NB: will need to expand on these
What are some device-related biofilm related infections?
Ventricular derivations
Contact lenses
Endotracheal tubes
Central vascular catheters
Prosthetic cardiac valvs, pacemakers and vascular grafts
Tissue fillers, breast implants
Peripheral vascular catheters
Urinary cathters
Orthopedic implants and prosthetic joints
What are some examples of biofilm caused tissue-related infections
Chronic otitis media, chronic sinusitis
Chronic tonsilitis, dental plaque, chronic laryngitis
Endocarditis
Lung infection in cystic fibrosis
Kidney stones
Biliary tract infections
Urinary tract infections
Osteomyelitis
Chronic wounds
Talk about the environmeent in the CF lung
Its is a heterogenous, hostile and stressuful environement, it is:
- osmotic
- oxidative
- nitrosative
- has sub-lethal concentrations of antibiotics
- presence of other organisms
- etc etc
Talk about the hypoxic conditions of the CF Lung
Low oxygen levels can influence oxidative stress
Under hypoxic conditions cells may rely more on anaerobic metabolism
- this is less efficient and more prone to generating free radicals which can lead to ROS formation
The CF lung is a hostile environment yet bacteria thrive here, why is this?
Due to evolutionary change in response to the selective forces that make the environment hostile
P. aeruginosa changes throughout an infection, what are some characteristic features of P. aeruginosa in early infection?
Non-mucoid phenotype
High virulence factors expression
Antibiotic sensitive
Regular metabolism
Normal mutation rates
What happens to P. aeruginosa as it changes from early infection to chronic infection?
Microoevoluion:
- Altered gene expression -> virulence factors such as the enzymes that are produced in early infection are down regulated
- selection of fiter variants
P. aeruginosa changes throughout an infection, what are some characteristic features of P. aeruginosa in chronic infection?
Mucoid phenotype
Biofilm growth
Reduced virulence factor expression
Auxotrophy
Antibiotic resistance
Adapted metabolism
Increased mutation rates
What happens in P. auriginosa chronic infection?
Frustrated phagocytosis
Tissue destruction
Inflammation
What three things are needed for the establishment of a chronic infection?
Biofilm Formation
Bacterterial diversity
Interbacterial communication
What is required for PA to form he initial monolayer of a biofilm
Flagella
LPS
OMPs?
What is needed for PA monolayer to develop into microcolonies?
Type IV Pili
Crc
algC increased
fliC decrease
What is needed for the PA microcolonies to develop into a mature biofilm
Acyl-HSLs
Alginate
What are the signals of the quarum sensing system?
Small extacellular signal molecules such as:
- homoserine lactones (HSLs)
- autoinducers (AIs)
Talk about quarum sensing when population density is low
Low cell population density
AIs are low in concentration
No biofilm formed
Talk about quorum sensing when population density is high
QS signal-receptor complexes form
Threshold AI concentration reached
Activation of transcriptiona regulators
Upregulation of biofilm genes
List some of the factors that P. aeruginosa QS regulates
Biofilm architecture
Elastase
Alkaline protease
Pyocyanin
Superoxide dismutase
Rhamnolipid
HCN
LasA
What is Pyocyanin of PA important for
Redox, physical and immunological effects
What is alkaline protease of PA responsible for
Cytokines
What is the role of superoxide dismutase of PA
Defence against ROS
What is the role of biofilm architecture in PA
Immune response evasion
antibiotic tolerance
dna secrtion
What is the role of LasA of PA
Competitiveness against staphylococcus
What is the role of HCN in P. aeruginosa
Impaired lung function
What is the role of rhamnolipid in PA
PMNs
Why are biofilm infections so difficult to treat
Treatment failure and infection recurrence is common in biofilms due to:
- resistance - bacteria can grow in the presence of an antibiotic
- tolerance - how to avoid antibiotic-induced cell death
Both tolerance and resistance result in biofilm recalcitrance
Biofilm tolerance is very different to classic resistance, talkabout detection of resistance with planktonic cells
Minimum inhibitory concentrations (MIC)
Minimum Bacteriocidal concetrations (MBC)
these are used to define the susceptibility breakpionts
PK/PD parameters that predict therapeutic success
Performed with plantonically growing bacteria
Biofilm tolerance is very different to classic resistance, talkabout detection of resistance with biofilm cells
Biofilm Inhibitory Concentration (BIC)
Minimum biofilm eradication concentration (MBEC)
Develop susceptibility tests specific to biofilm-growing bacteria
What three mechanisms contribute to biofilm tolerance to antibiotics
Phyical:
- the matrix - its 3D structure
Differential physiological activity:
- heterogeneity of microbial biofilms
- persister cells
- small colony variants
Adaptive Responses
In what four ways can the matrix 3D structure provide physical tolerance to antibiotics
Antimicrobial penetration
Hyper production of alginate
Filamentous phages slow down antibiotic diffusion
Biofilm regarded as independent pharacokenetic microcompartment
How can the matrix 3D structure prevent antimicrobial penetration?
Reduced or delayed penetration ECM
Antimicrobials binding ot matrix xomponents
Tobramycin and colistin cationic fail to penetrate due to binding to polysachharide and eDNA
But the negative charged ciprofloxacin pentrates well
How can hyper production of alginate contribute to antibiotic tolerance
Mucoid strains are more resistant to tobramycin
Cationic antimicrobials bind to negatively charged alginate
How can filamentous phages slow down antibiotic diffusion?
Pf phages carry a negative charge which facilitated binding, sequstration and tolerance to any cationic aminoglycosides and antimicobrial peptides
How can the 3D matrix of biofilm and its independent pharacokenetic microcompartment contribute to resistance?
Difficult to reach wih systemicallically admiinisted antimicrobials
Hard for antimicrobials to get through etc
What two factors of biofilm contribute to physiologica tolerance
Stratified baterial physiology in biofilms contribute to:
- Altered growth rate
- Hypoxic Confitions
Talk about the altered grwoth rate of biofilms and how this contributes to physiological tolerance towards antibiotics
Reduced metabolic activity of bacteria in the biofilm
Antibiotics usually target actively dividing cells
At least two sub populations:
- Aerobic
- Anaerobic
Talk about the hypoxic vonditions of biofilms and how this contributes to antimicrobial tolerance
Aminoglycosides, fluoroquinolones, B-lactams dont function well in these conditions
Collistin is effective though and can kill the inner part of the biofilm
Which antibiotics are and arent affective against biofilms
Tetracycline can only kill outside cells o bifilm
Ciprofloxacin can kill outside cells and some inside cells
Collistin can kill internal cells but not external
What are persister cells in biofilm
Subpopulation of tolerant/dormant bacteria
They usually only account for <0.1% of cells
Phenotypic switch (not genetic change) whereby bacteria differentiate into a dormant state
These cells lack genetic resistant mechanisms
They are non dividing cells that can revert to ast dividing cells
NB: Suggestions that they are responsible for biofilm regrowth following biocide exposure
What are small colony variants of P. aeruginosa?
Phenotypic variants of P. aeruginosa
Theyre a strategy for bacterial surival uner various stress conditions
They are good biofilm formers
Theyre associated with persistance in host cells and tissues
Theyre less susceptible to antibiotics than the wild type
Theyre directly associated with antibiotic toleranc and persistant infections
Why are P. aeruinosa SCV better biofilm formers than wild type?
Upregulation of cyclic diguanosine-5’monophosphate (c-di-GMP)
Exopolysaccharide production
Auto-aggregation
Slow growth rates
What are small rough colony variants of P. aeruginosa
Small slow subpopulations
They account for 3% of P. aeruginosa positive sputum
The recovery of SCV correlates with parameters revealing poor lung function and an inhalative antimicrobial therapy
Give a common example of a SCV
Rugose SCVs ‘Winkly Spreaders’
Mucoid phenotype is also an example of a phenotypic change
Talk about the SCV Wrinkly Spreaders
WSP phenotype
Increase expression of psl and pel synthesis
Increases c-di GMP
Decreased motility
Often triggered by exposure to different antimicobials such as tobramycin
Talk about how biofilm induces antimicrobial tolerance through adaptive tolerance
Exposure to antimicrobials can induce transient resistant phenotypes through adaptive resistance
Acquired by sub populations of microorganisms in biofilms
Dependent on presence of antimicrobial agents:
- due to the rapid uprgulation of resistance genes
- these genes are no longer expressed when the antibitoic is no longer present
Adaptive tolerance can be non specific or specific for a particular class of antimicrobial
Give an example of adaptive tolerance?
Upregulation of efflux pumps
Upregulation of effluc pumps is an example of what kind of adaptive tolerance?
Non-specific adaptive tolerance
Talk about upregulation of efflux pumps as an example of non-specific adaptive tolerance
Upregulation of MexAB-OprM efflux pumps
These span the outer membrane and activey efflux lipophilic and amphiphilic drugs
They can pump out multiple classes of antimicrobials
Multiple efflux MEX provides tolerance against what?
Against several classes including azithromycin and collistin
What triggers Multiple efflux MEX
Triggered by the presence of antimicrobials
Its production is a bacterial stress response
e.g. it can occur due to oxidative stress in the CF lung environment
Give some examples of specific adaptive tolerance in biofilms?
B lactamase production
Synthesis of periplasmic glucans - tobramycin resistance
alteration to LPS in the presence of colistin
Talk about B lactamase transcription in response to B lactam antibiotics as a form of specific adaptive tolerance
Induction of B lactamase transcription in response to B lactam antibiotics e.g. imipenem
Induces high levels of B lactamase in the biofilm
Changed the PKPD
Time dependent to concentration dependent
Talk about specific adaptive tolerance to tobramycin in biofilms
Synthesis of periplasmic glucans
Mediated by the ndvB gene
It is strain specific i.e. we dont see it in all PA isolates
It binds tobramycin thus preventing cell death by sequestering the antibiotic molecules away from their cellular targets - 30S as its an aminoglycoside
ndvB mutants can still form biofilm but lack resistance to Tobramycin
When grown planktonically the ndvB mutant and wild type were equally susceptible to the aminoglycosides
What gene is responsible for Tobramycin resistance in PA
ndvB
Talk about colistin resistance as an exaple of specific adaptive tolerance in biofilm
Alteration to LPS in the presence of colistin
The “cap” component of typica PA biofilm exhibits tolerance to colistin
Exposure to colistin triggers LPS modification
It results in the reduction in the negative charge of the LPS
This reduces the binding of the cationic colistin (+ charge) to the bacterial cell surface of the P. aeruginosa
How exactly is the LPS modified during adaptive tolerance against colistin in PA biofilm
upregulation of the two-component regulatory system pmr
This regulates the arn genes
This results in the addition of a cationic (+) sugar amino arabinose to the lipid A phosphate group of the LPS
This reduces the negative charge of the LPS thus reducing the binding of cationic colistin to the bacterial cell surace of PA
What are the methods of biofilm-associated tolerance against beta-lactams
Restricted penetration (inactivation of B-lactam molecules by B-lacamase in the biofilm matrix)
What are the methods of biofilm-associated tolerance against collistin only
Effllux pumps (MexAB0OprM, MexCD-OprJ)
What are the methods of biofilm-associated tolerance against aminoglycosides only
ndvB
brIR
PA1874-1877
What are the methods of biofilm-associated tolerance against beta-lactams, fluoroquinolones and aminoglycosides?
Low O2 concentration
Low metabolic activity
Stringent and SOS response
Persisters
Efflux pumps
What are the methods of biofilm-associated tolerance against aminoglycosides and colistin
pmr/arn operon
Restricted penetration (binding to components of the matrix: eDNA, polysaccharides)
How does multi-species biofilm affect resistance
Multi-species are less suceptible than mono-species biofilms
Bacterial species within a complex biofilm have been shown to protect susceptible species
Give an example of a multi-species biofilm
A Bacillus subtilis endoscope washer disinfector isolate
A strong EPS producer resistant to chlorine (0.03%) hydrogen peroxide (7.5%) and peraetic acid (2.25%)
The B. subtilis biofilm was able to protect S. aureus from the peracetic acid of 0.35%
Acinetobacter johnsonii was shown to protect salmonella enterica Liverpool in a dual biofilm against benzalkonium chloride (300mg/L)
What impact do biofilms have on disinfectant efficacy?
Failure of surface disinfection
Bacteria remain on surfaces even after biocide exposure
Susceptibility of biofilms to disinfction
Lack of disinfectant efficacy was associated with:
- biofilm thickness
- biofilm maturity
- presence of persister cells
What challeges are associated with biofilm in the health care environment?
Cleaning and disinfection in the Health Care environemen poses a serious challenge
Cleaning is significantly more complex by the presenc of biofilms
Think drain biofilms, dry surface biofilms, medical device biofilms
Give some examples of wet biofilms that pose a problem in healthcare settings
Drains
Sinks
Showers
Talk about dry surface biofilms
Relatively new phenomenom only discovered within the last decade
Can grow on dry furnishings
These tend to be much more resistant to disinfectants
Talk about drain biofilms
Hydrated biofilms
Sinks, taps, drains, showers, u bends etc
Constantly hydrated biofilms -> nutrients supplied by pouring non water substances such as coffee down sinks etc
Oftwn grow in humid conditions
Well protected environments - cannot be easily cleaned
Several protocols and products to decontaminate sinks and drainage systems but biofilm regrowth occurs quickly after treatment - disinfection only works short term
What organisms are we concerned with in drian biofilms
There is a colonisation rate of>40% in ICU sinks
CPEs and P. aeruginosa are of most concern
How common is biofilm drain contamination
Colonisation rate of >40% in ICU sinks
What are dry surface biofilms?
Biofilms present on dry surfaces often embedded in gloopy EPS, often subjected to repeated desiccation periods i.e. not just planktonic cells - can regrow etc
Often multi-species to offer extra protection
Widespread in the healthcare environment
Can by found on tray tables, mattress, pt folders, keyboards etc
They cannot be recovered by swabbing
They are less susceptible to phsyical and chemical disinfection/Sterilisation - much more resistant
How common are dry durface biofilms
Recent study revelaed 90% of surfaces smapled harboured a DSB
What is the only way to recover Dry surface biofilms, what is the issue with this
They arent picked up by routine swabbing
They have to first be exposed to disinfectants in order to be able to pick them up on a swab
This causes problems when surfaces are wiped with disinfectants as staff and patients can ow pick them up etc
What is the most common DSB
S. aureus DSB
What is currently the most effective way of removing S. aureus DSB
Using biocides such as benzalkonium chloride (<0.5%), peraetic acid (250 ppm), NaOCl (1000ppm) in combination with wiping reduced effectively
There was a greater than >4log10 reduction in S, aureus DSB
What is the main issue with transmission of DSBs
There is an issue with bacterial transfer from DSB post disinfection wiping:
- only a couple of commerically available products prevented bacterial transfer ie. direct transfer or transfer via gloves
Explain using an example just how resistant a S. aureus DSB can be
Some S. aureus in DSBs can survive exposure to 20,000ppm chlorine which prouced a >7 log10 reduction in viability and a reduced biofilm biomass of >95%
However still viable S. aureus were able to regrow
Expoure to peracetic acid or chlorine were not efficacious in produceing a <3 log reduction in viability in the absense of soiling
Hydrogen peroxide has no activity against the biofilm at all
What is the most common chlorine product used
Chlorclean
What is the most common peracetic acid product used?
Proxitane
What is the most common hydrogen peroxide product used
Oxivir
When trying to remove both drain biofilms and dry surfae bioilms, what method are we trying to mimic
Trying to mimic autoclaving
How does heat disinfection affect drain biofilms
Moist heat disinfection at 121 defrees for 30mins (autoclaving)
-> culture negative and no recovery
Dry heat sterilisation at 121 degrees for 20 minutes
- no growth
How does heat disinfection affect dry surface biofilms
Moist heat disinfection at 121 degrees for 30 minutes:
- culture negative but did recover
Dry heat sterilisation at 121 degrees for 20 minutes:
- Only a <2 log10 reduction in viability
Talk about medical device biofilms
biofilms on medica devices and implants
They increase the risk of infection
They are often challenging to remove as theyre oten a combination of hydrated and DSBs
what medical devices are most often coated in biofilms and why?
Theyre a shared, multi-use piece of equipment
Small channel diameters of endoscopes make cleaning difficult
Biofilms easily form on endoscope lumens after squential hydration and dehydration phases
Pseudomonas is most common
What bacteria is most commonly associated with endoscopes
Pseudomonas aeruginosa
Talk about the disinfecion of P. aeruginosa biofilms on medical devices
Even with treatment of 4000ppm of peracetic acid P. aeruginosa will survive - residual amount remains
Some studies show that paracetic acid is affective in biofilm removal but when the drying process after disingection was missed then regrowth occurred within 48 hours
What chalenges do we face with biofilms in terms of infection prevention and control
Hydrated biofilms: lack of disinfectant efficacy and biofilm regrowth is inevitable
DSB may promote prolonged pathogen survival
Reservoirs prove a constant issue
Medical devices require high level disinfection
Why are biofilm reservoirs such an issue in infection precention and control?
They may need mechanical removal of bifilms
Often result in transferability - much more likely to transfer after wiping etc
Disinfectant wipes not efficeint
One wipe one surface one direction is vital to prevent spread of DSBs - need to educate staff etc
Why are medical devices such a challenge with infection prevention and control?
High-level disinfection is part of the reprocessing procedure
Biofilms suggest suboptimal cleaning/disinfection protocols
Combo of wet and dry can be challenging
Talk about the current scene of biofilm therapeutics
There is a mismatch between curent antimicrobials and anti-biofilm activity
Have to provide high antibiotic concentrations through topical administration
Combined and sequential antimicrobial therapies
AST in lab doesnt acuratly reflect what happens in vivo - clinicians often treat emperically
Must rethink what is the optimal strategy to fight chronic biofilm
Antibiotic treatment strategies for combining biofilm infections
Currenly how are CF exacerbations treated?
antimicrobial therapy to reduce bacterial load and stabilise lung function
Dual therapy with a B-lactam and an aminoglycoside is standard
Most decisions are empirical based on patients preivous responses, age, colonising organisms and degree of exacerbations
Antibiogram is not considered - ast not reflective - poor predictor of clinical response
Currently what AST is done for CF
Ast is not recommended
Standard susceptibility testing methodologies only provide relevant information for the planktonic component against a single antimicrobial
AST will not eradicate the tolerant subpopulation of sessile/persister cells
How representative is a CF sputum, BAL or cough swab?
The same PSAE morphotype gave different sens patterns
Number of PSAE picked (mean=4) differed depending on the scientist, the lab and the time
Isolates of PSAE where indistinguishable by typing but gave different sensitivity pattenrs
Multiple comination bacteriocidal testing MCBT testing did not result in beter clinical and bacteriological outcomes compared with therapy directed by standard culture and sensitivty techniques
What are the requirements for antimicrobial biofilm susceptibility testing?
Need to grow non motile organism in close proximity, need to provide a surface for attachment and allow quarum sensing
Require a procedure to allow addition of antimicrobials, single and multiple after biofilm formation
Need a method to detect the effcicay of those antibiotics that correlates with the in vivo response
Provide a reproducible, standardised, useful clinical information e.g. BIC, MBEC etc
Needs to be cheap, usable and easy to set up
What are the two different types of biofilm assays available today?
Those that generate biofilms on microtitre plates
Those that generate biofilms on different surfaces such as indwelling devices, lines and catheters
List some examples of AST biofilm methods that generate biofilms on microtitre plates
The Calgary device (MBEC assay)
Poloxamer gel method
Crystal violet method
Microplate alamar blue assay (MABA)
Others: XTT, Syto9, FDA, DMMB, MUG
Give an example of an assay for biofilm AST based on a method of generating biofilms on different surfaces such as indwelling devices ,lines etc
Biofilm disk reactor system (modified Robbins device)
What was the historic crude methof od biofilm AST
Crystal violet + spectrophotometer
Calgary method is preferred
What was the historic crude methof od biofilm AST
Crystal violet + spectrophotometer
Calgary method is preferred
What biofilm AST methods are available in labs today
None, they are all research based atm
What is the MBEC method of AST for biofilms
The calgary device assay which involves generating biofilms on plastic pegs attached to lid of 96 well microtitre plate, plate is rocked to induce sheer forces - biofilm is transfered from pegs by sonication
You can challenge the growth of the film by introducing any antibiotic you want into the well
What are the benefits of the MBEC assay
Can vary concentration
Can select specific antibiotics
In general what do MBEC results reveal about resistance in biofilm compared to planktonic cells
There were really significant differences in the MIC required to remove biofilm
Aztreonam, ceftazidime, imipenem and piperacillin showed no activity at >1000 fold the MIC
Gentamicin required to eliminate the biofilm was 60 fold greater than the MIC
Tobramycin and amikacin were effective against the biofilm wihin the susceptible range for the MIC assay
Ciprofloxacin required a 4 ug/ml to eradicate the bofilm but theis falls outside the susceptible range by MIC standards but still represents obtainable drug levels
Talk about MBEC results for imipenem and colistin
Following results are from a mouse lung model:
Its difficult to reach the MBEC of colistin and imipenem in vivo without causing side effects and toxicity
Thus biofilm eradication seems impossible using antibiotic monotherapy via systemic administration - could never get a concentration high enough
NB: clinical target of drugs is different for biofilm than planktonic cell infection
What happens when you use the dosage recommended from planktonic MIC on a biofilm
Reduction in the size of the biofilm embedded population but the infection will persist
The bacteria will resume growth after treatment ends
What are the four anti-biofilm strategies
Prevention
Weakening
Disruption
Killin
How do we prevent biofilms
Antibiotic prophhylaxis
Targeting of surface molecules
Targeting c-di-GMP signalling
How do we weaken biofilms
Inhibition of efflux pumps
Degradation of extracellular matrix
Targeting of extracellulae and intracellular signalling molecules
How do we disrupt biofilms
Mechanical disruption
Biological disruption with enzymes
Targeting of extracellular anf intracellular signalling molecules
How do we kill biofilms
PK/PD guided antibiotic treatment
Targeting subpopulations with different classes of antibiotics
Targeting of the bacerial membrane
What are the four categories of novel anti-biofilm strategies
Reversible-irreversible attachment
Microcolony formation
Biofilm maturation
Dispersal
Give some examples of reversible-irreversible attachment anti-biofilm methods
Antiadhesion agents such as mannosides, pilicides and curlicides in inhibition of UPEC biofilms
Antibiofilm polysaccharides
Signal transduction interference
Give some examples of microcolony formation anti-biofilm methods
Lytic phages
Silver nanoparticles (silver has antimicrobial properties)
EPS-degrading enzymes
Antimicrobial peptides
Antibiofilm polysaccharides
Signal transduction interference
DNAse I, Dispersin B (breaks down extracellular DNA in biofilm)
Chelating agents
Give some examples of biofilm maturation anti-biofilm methods
Exact same as for microcolony formation
Give some examples of dispersal anti-biofilm methods
c-di-GMP enginerring to promote motility versus sessility
Introduction of dispersing signals e.g. n-amino acids/nonpermidine in the case of B. subtilis
Talk about the use of silver in biofilm treatment
Silver + antibiotics are used to kill persister cells
Talk about QS inhibitory in biofilm treatment
QS inhiitors jam communications needed to form both microcolonies and mature biofilms
Give 4 examples of methods that stop biofilm forming
c-di GMP inhibitors
QS inhibitors
Lactoferrin
Biosurfactants
Honey
Non pathogenic bacterial colonisation
Vaccination
Talk about lactoferrin on biofilms
An innate glycoprotein with broad actibacterial and antibiofilm properties
Talk about biosurfactants on biofilm
Biosurfactants such as rhamnolipids, sophorolipids and lipopetides act as anti-biofilm agents with an inhibitory effect
Give four exampls of methods that kill biofilms
Lytic bacteriophages
Silver+antibiotics
ADEP4 + rifampicin to kill persistor cells
Honey
Talk about honey and biofilms
Honeys bioactive components inhibit biofilm formation and quorum sensing activities
Honey has antibacterial activity even against MDR bacteria
Honey has antibacterial agents which can destroy bacterial cells through different mechanisms
Talk about ADEP4 and rifampicin to kill biofilms
Acyldepsipeptide 4
- an exploratory antibiotic with a novel mechanism of action which activates the ClpP protease and casuses cells to seld digest
Combination with rifampicin leads to eradication of persisters, stationary and biofilm populations (study in mice)
Talk about ADEP4 and rifampicin to kill biofilms
Acyldepsipeptide 4
- an exploratory antibiotic with a novel mechanism of action which activates the ClpP protease and casuses cells to seld digest
Combination with rifampicin leads to eradication of persisters, stationary and biofilm populations (study in mice)
