-organized communities of bacteria that are adhered to a surface and surrounded by (enmeshed in) matrix of extracellular polymeric substances (EPS)
-composed of single bacterial species
-biofilm formation occurs as a result of coordinated chemical signaling between cells (quorum sensing)
-when increased amounts of bacteria are present in close proximity to each other → autoinducer concentration increases
once binding of autoinducer molecules has exceeded a certain threshold → signaling cascade initiated that modulates gene expression → modulates bacterial physiology to encourage community development
-bacteria in biofilm behave as a group
sense and respond to stimuli in a coordinated manner
-some biofilm structures include network of pores, held together by EPS
provides a primitive circulatory system
O2 and nutrients, bacteria deep inside biofilm have different physiology than bacteria closer to surface
-physical biofilm formation process
adherence of bacteria to a surface
growth & production of EPS, resulting in much stronger (often irreversible) attachment leading to biofilm maturation
dispersion of single cells (or parts of the biofilm) from the site of mature biofilm
Extracellular Polymeric Substances (EPS)
slimy, film-like substance produced by bacteria in biofilm
composed of polysaccharides, proteins, nucleic acids
Quorum Sensing
uses signaling molecules (autoinducers) produced by bacteria
bacteria has receptors for these signaling molecules
Planktonic
single bacterial cells that are growing in liquid culture
single bacterial cells swimming freely above a film
can be recruited to a biofilm or may have seeded off an existing biofilm
-commonly found environmentally and with biological hosts
classified as benign or pathogenic
Benign Environmental Biofilms
don’t cause human disease
responsible for significant industrial problems (corrosion)
ex: slipper coating on rocks in streams, coatings on ship hulls
may also impact human health
ex:
colonization of hot water systems in hospitals by mycobacterium avium
drinking water reservoirs contaminated by salmonella typhimurium
can attract, recruit and concentrate pathogenic bacteria that might not form a biofilm on their own
Pathogenic Biofilms on Medical Implants
Gram (-) and Gram (+) readily form biofilms on many foreign devices implanted into human bodies
account for significant human infection & disease
ex: affected medical devices (urinary catheters, heart valve implants, hemodialysis equipment, dental implants)
may also form on human body surfaces
ideal environment for formation of different classes of biofilms
many of these infections caused by opportunistic pathogens that are human commensals
biofilm infections result in chronic disease difficult to treat with antibiotics
ex:
cystic fibrosis (CF) infections in lung caused by pseudomonas aeruginosa
tuberculosis infections caused by mycobacterium tuberculosis
urinary tract infection caused by escherichia coli
ear infections (variety of bacteria)
tonsillitis (variety of bacteria)
oral films
primary causative agents of dental caries and gingivitis
-biofilm communities significantly more resistant to antibiotics and antimicrobial stressors
including those mounted by natural host responses (immune system) compared to planktonic bacteria of same species
EPS Matrix
complex EPS layer enmeshing the biofilm can impede penetration of antimicrobial agents to bacteria buried in depths of biofilm
Nutrient and O2
bacteria within a biofilm are subject to nutrient and O2 gradients
bacteria closer to surface have better access to nutrients and O2 than interior bacteria
bacteria in biofilm interior (while alive) are metabolically inactive
antibiotics most effective against metabolically active cells
bacteria in interior protected
Persister Cells
small percentage of population remains viable despite prolonged exposure to antimicrobial or even increased dosage of antibiotics
confer no heritable resistance to progeny once selective pressure is removed
able to survive for extremely long periods of time
-interfering with EPS synthesis
coating medical devices with chemicals that hinder matrix formation
-inhibiting adherence of biofilms to their surface substrate
identifying chemicals that bind to bacterial cell surfaces prevent biofilm formation
-targeting autoinducers
bacteria unable to signal to each other → unable to form/maintain a biofilm
-bacteria rely on signal transduction systems in order to form biofilms
senses environmental changes
-during biofilm formation, two core signal transduction systems play collective role in mediating biofilm formation
quorum sensing
chemotaxis
-prokaryotes able to sense presence of others of their own kid that are nearby in environment
-type of environmentally-regulated gene expression where it’s regulated by population density
prokaryotes sense presence of their own kind and work together
large changes in gene expression change a cell’s phenotype from individualistic growth to community-based growth
-used to ensure sufficient numbers of a given species are present before initiating a response that requires certain population density to have an effect
-several different quorum sensing mechanisms of varying complexity based on either one-component or two-component systems have evolved in prokaryotes to regulate gene expression in response to population density
-involves a communication chemical autoinducer (AI)
-Lsr quorum sensing system
-LsrR
global regulator of system in an inhibitor of its own operon and lsrABCD operon
keeps quorum sensing system shut off when cell concentration is low
represses expression of both operons which includes its own expression
-one operon contains lsrK and lsrR genes that encode 2 regulators in the system
-second operon consists of several genes lsrABCD that encode proteins involved in sensing and transporting autoinducer into cell when it reaches the threshold
-LuxS enzyme responsible for synthesizing autoinducer (A12)
-converts a molecule (DPD) into AI2 which is transported out of cell by membrane-bound protein (YdgG)
-even when population density is low → cell continues to secrete AI2 to indicate to other cells that it’s present
-as population density increases → concentration of AI2 increases as well
-once concentration reaches threshold → LsrABCD complex (membrane bound) starts to import AI2 from environment into cell where LsrB senses external AI2 levels
-after AI2 is imported into cell → AI2 phosphorylated by LsrK → AI2 + P binds to LsrR and relieves repression of 2 lsr operons to allow further production of quorum sensing proteins
-LsrR
global regulator
regulates its own operon and other genes in cell including other regulators
able to activate a cascade of changes in gene expression in cell
changes often result in cell undergoing some form of community based phenotype
can act as transcriptional repressor or activator
binds to AI2 + P to change how it binds to certain promoters
-biofilms are bacteria’s response to persistent environmental changes leading individual bacteria to aggregate in large microbial communities of immobile cells
-requires transformation from planktonic bacterial lifestyle
requires motility to respond to rapid fluctuations in their microenvironments
responsive and motile lifestyle dependent on chemotaxis
-chemotaxis is a change in motile behaviour as a result of sensing a chemical concentration difference (concentration gradient) in the environment
immediate change in behaviour doesn’t require synthesis of new proteins
Positive Chemotaxis
movement toward higher concentrations of chemical substances (attractants)
Negative Chemotaxis
movement away from higher concentrations of chemical substances (repellents)
-in order to exhibit chemotaxis, organisms must be able to sense when environmental concentration of an attractant or repellant is increasing or decreasing and be able to respond to the environmental sensation appropriately
Spatial Sensing
concentration difference sensed by comparing concentration of chemical at two points in space at time using receptors at the front and back of the cell
Temporal Sensing
concentration difference sensed by comparing concentration of chemical at two points in time
Run
smooth forward motion of swimming organism
typically associated with flagellar rotation in counter-clockwise rotation
relate flagellum to tail-like structure
Monotrichous Organisms
single flagellum organisms
change direction by reversing flagellar rotation (clockwise)
pulls cell backward in erratic way causing random change in direction for subsequent run to twitching motion (tumbling)
others change direction by briefly stopping flagellar rotation
random motion points them in random direction for a subsequent run
Peritrichous/Lophotrichous Organisms
multi flagella scattered over cell surface
lophotrichous organisms possess a tuft of flagella at a single location
rotation of flagella in one direction causes them to come together in a bundle and act in coordinated manner → running motion
tumble results from a switch in direction of rotation by at least one flagellum
bundle “flys apart” → run disrupted → bacterium undergoes chaotic 3D whirling motions (tumble)
subsequent run occurs in random direction
-in absence of chemical concentration gradient → motility rarely results in net movement in any particular direction
-cells follow random pattern (Brownian motion)
no set end point or net directionality
-depending ont he chemical and chemical gradient present → chemoattractant if promotes movement
cells to move away from it → chemorepellent
-relative position and type of chemical may promote either tumbling or running
-can’t say that a single type of chemical only promotes one type of reaction
dependent on the location
-if chemoattractant behind cell → may tumble first to change direction then run towards it
-if senses chemoattractant in front of cell → run towards it
Chemoreceptors
proteins clustered at the front of the cell
bundled in high concentrations in order to be more sensitive to signals and promote signal amplification
-if chemorepellent (ex. antibiotic) is sensed in front of the cell → cell will tumble and change direction
a class of chemoreceptors will sense signal and activate CheA (response regulators in pathway)
once activated by chemoreceptors → CheA phosphorylates CheB to activate enzymatic activity
CheB demethylates CheA (protein that just activated it)
when CheA is demethylated → able to phosphorylate second protein CheY
when CheY phosphorylated → changes conformation so it actively binds to flagella motor switching its default, unbound rotation from counter-clockwise (CCW) to clockwise (CW) causing cell to tumble and change direction
CheA
kinase; enzyme capable of phosphorylating other proteins
CheB
demethylase; enzyme that removes methyl groups from other proteins
-cell has changed directions and now wants to put repellent behind it and run
-resetting system means going back to default rotation of flagella when it’s not bound to CheY-P which is counter clockwise (CCW)
cell senses it’s facing direction where antibiotic concentration is lower
CheA stops phosphorylating CheB
CheR (methylase) starts to methylate CheA to CheA-CH3
CheA-CH3’s kinase activity is turned off → can’t phosphorylate CheY
CheZ dephosphorylates CheY to change its conformation → can’t bind flagellar motor
free, unbound motor rotates in CCW direction to promote running