Biofilms test 1

Infectious kidney stones are composed of:

-Bacteria -Bacterial exo-products -Mineralized stone material

characteristics of biofilms:

-Complex, dynamic communities -Heterogeneous, often multi species -Exhibit altered gene expression when compared to their planktonic counterparts -Coordination of behavior through quorum sensing mechanisms -Less susceptible to antibiotics and other environmental stressors

what are some examples of biofilms formed on medical devices?

-In dwelling catheters -prosthetic devices -dressings -implants -tympansostomy tubes (prevents fluid buildup in ear) -pacemakers

Bacillus subtilis

-gram positive, rod shaped, commonly associated with soil, human skin, and human gut colonization. -True commensal organism: not an opportunistic pathogen, and can often outcompete invading pathogens. -Great model organism

Myxococcus xanthus

-gram-negative, rod shaped bacterium -normally exists as a single species biofilm (termed a swarm) -model organism for community behavior -their communities switch between swarms and fruiting bodies

amyloid biofilm (S. aureus)

-have surfactant qualities that help with swarming -amyloid aggregates promote biofilm formation -amyloid proteins were important in our evolution from single to multicellular organisms

how are localized gradients a protective property in biofilms?

-heterogeneity results in better bacterial defenses -the altered micro environments are creating different gene expression profiles

what are advantages and disadvantages of high alginate production

-high alginate production can protect against host defenses and antibiotic treatment -high alginate production is metabolically expensive

nutrients scavenged from water in biofilms

-inorganic particles (clay, salt, hydroxides, nano-particles) -organic particles (humic substances, debris, cellulose, nano particles -metal ions -organic molecules (polar and non polar)

Amyloid biofilm of S. aureus

-mediated by phenol-soluable modulins (PSMs) -very hydrophobic and very hydrophillic sides -important virulence factors because they form pores that can lyse cells (this is why they are very important virulence factors)

requirements for biofilm formation

-microbes -moisture (fresh water, salt water, oil pipelines, in the human body) -surface (rocks, countertops, human tissue, etc.) -nutrients (certain biofilms can even thrive on petroleum oil) anytime these 4 ingredients are present, a biofilm can and usually will form

step 1: reversible attachment

-often thought to require the presence of a "conditioning film"

Antibiotic tolerance

-phenotypic changes (growing differently) -sessile cells are metabolically inactive -transient tolerance

extracellular matrix composition:

-polysaccharides -proteins -DNA -lipids

Life cycle of M. xanthus

-starvation response: flee or form a fruiting body -purpose of forming a fruiting body is to generate myxospores -myxospores are resistant to environment but not as tough as endospores -process driving this differentiation is not as well studied as in B. subtilis

life cycle of M. xanthus

-starvation response: flee or form a fruiting body -purpose of forming a fruiting body is to generate myxospores -myxospores are resistant to the environment but not as tough as endospores -process driving this differentiation is not as well studied as in B. subtilis

Sites of primary biofilm infection

-subvenous catheter -mouth -artificial hip implant -urinary catherters and other implantable devices

characteristics of biofilm infections

1) Form preferentially on foreign bodies, dead or damaged tissue 2) Slow to develop 3)Respond poorly or only temporarily to antibiotics 4) Collateral damage to neighboring healthy tissue

3 major types of cell differentiation

1) Physiological adaptation: gene expression changes in response to altered microenvironments 2) Mutation: genotypic variation and natural selection can lead to propagation of mutants with greater fitness 3) Stochastic gene switching: random "ON" or "OFF" toggling of alternate gene programs

1)Why would being in a biofilm affect survival in nutrient-deplete conditions? 2)How are cells in a biofilm more protected from harsh environments?

1) The EPS has nutrient scavenging properties -water channels -negatively charged, they can get positively charged ions, and can dissolve organic carbon compounds 2) -The EPS is thick -decreased phagocyte penetration -degraded antibiotics -decreased metabolism -quorum sensing mechanisms

what makes a great model organism in biofilm formation?

1) genetically tractable 2) easily grown 3) great biofilm former 4) perfect model for "bet hedging"

biofilms in nature can be 1) 2)

1) natural, potentially beneficial sources of microbial diversity 2) harmful contaminates impacting health and industry

Steps of biofilm growth and development

1) reversible attachment 2) irreversible attachment 3) microcolony formation 4) maturation 5) detachment

what are the modes of dispersal (step 5 in biofilm formation)

1) sheer force flow: lotic environments 2) passive dispersal: dispersal driven by the environment instead of by the biofilm 3) active dispersal: dispersal of biofilm driven by self

B. subtilis bet hedging strategies in times of stress: Matrix production

Matrix production: -Pro: enables biofilm formation -Con: Highly energetically expensive

step 4 in biofilm formation

Maturation -the mature biofilm is shaped by the environment and by the genetics of the microorganism -the same microorganism can form different types of mature biofilms in different environments

Biofilms play a role in the following infections:

Otitis media Chronic wound infection Recurrent tonsilitis Urinary tract infection Periodontal disease Chronic lung disease Infectious kidney stones Bacterial endocarditis Osteomyelitis

common causes of post operative wound infection

P. aeruginosa (64%)

Amyloid biofilm of S. aureus

PSMs (phenol-soluable modulins) -these are important virulence factors -lyse host cells -have surface qualities that can promote biofilm dispersal and spreading of epithelial surfaces -can also accumulate as amyloid aggregates that promote biofilm development -60% of s. aureus secreted biomass

what does Pel EPS do in P. aeruginosa

Pel promotes cell-to-cell interactions and protects against aminoglycoside antibiotics

Microbes associated with infections of the cystic fibrosis

Pseudomonas aeruginosa Staphylococcus aureus Haemophilus influenza Burkholderia spp.

what does Psl EPS do in P. aeruginosa

Psl promotes resistance to polysorbate 80 (biofilm inhibitor)

what is a biofilm?

a community of microorganisms that is attached either to a surface or to each other and is encased in a matrix (EPS- extracellular polymeric substance)

what is a conditioning film?

a conditioning film is the deposit (or aggregation) of macromolecules such as proteins and polysaccharides onto a surface (a conditioning film can form even in a sterile environment)

what is N-acetylglucosamine? (NAG)

a derivative of glucose as a biopolymer, it is an important component of cell walls. Component of the polysaccharide biofilm in S. aureus.

Infectious kidney stones

a true biofilm associated infection -kidney stone + UTI -bacteria remain viable after antibiotic administration

role of cells - yeast

adherence: these cells colonize surfaces to form the initial biofilms Dissemination: this cell type can more easily move to other locations (for example, these cell types are more adept at traveling through the bloodstream)

what is the algL gene do?

alginate lyase gene, this is involved in alginate export and degradation. if there is a deletion mutation in this gene, you will end up with a "mucoidy" biofilm

what is mucoidy EPS caused by?

alginate overproduction, deletion of the alginate lyase gene (algL)

A group of cells in an anoxic portion of the biofilm have switched off their respiration machinery and are no longer generating a PMF (proton motor force). They survive a treatment with the antibiotic tobramycin which requires a PMF to enter the cell. This is an example of

antibiotic tolerance

"mucoidy" P. aeruginosa

becomes important during chronic infection -mucoidy is due to the overexpresssion of the 3rd type of EPS, alginate. -happens if the algL gene is deleted

microbial cities: benefits for bacteria to stay/leave

benefits to stay: biofilm formation assists in feeding microorganisms. water channels form in the EPS to help feed the biofilm benefits to leave: competition

why are biofilms often formed in response to nutrient starvation?

biofilm formation assists in "feeding" microorganisms under nutrient starved conditions, dispersal must be great

biofilm maturity results in multicellularity which results in:

biofilm multicellularity results in better bacterial defenses -nutrient depletion creates zones of altered activity -outer layers of biofilm can absorb cell damage -inner layers of biofilm cells have more time to initiate stress response -"persister" cells may be present in higher numbers

what was the earliest form of microbial life observed?

biofilms (leewenhoek observed dental plaque on teeth)

do the nutrient acquisition properties of a biofilm eliminate any protective effects of the biofilm?

biofilms are 1000x less susceptible to antibiotics than their planktonic counterparts

what is secondary site of biofilm infection?

biofilms can travel from the primary sites of infection where it was established to a secondary site through the bloodstream

many nosocomial infections are a result of__________

biofilms formed on medical devices

cyclic-di-GMP phosphodiesterase

breaks down c-di-GMP. cells that lack this enzyme have too much c-di-GMP and are hyper biofilm formers.

what happens if an overexpression of autolysins occurs?

can lead to self destruct mode and release of extracellular DNA, this becomes a community resource for neighboring cells.

what are autolysins used for?

cell wall restructuring to allow these cells to grow and ultimately divide into two cells, these are very important for the survival of cells. used usually for cell growth

why are biofilms in infection so dangerous?

cells in infected sites experience many of the biofilm-associated protections seen in laboratory biofilms. -Biofilm associated infections are slow to develop and persist for a long time -biofilm associated infections respond poorly to antibiotics -chronic inflammation assists in biofilm formation

coagulase negative species such as S. epidermidis tend to be?

commensuals

B. subtilis bet hedging strategies:

competence sporulation matrix production endospore formation

what are the microbial warfare strategies used by microorganisms to defeat competitors?

competition between cells in biofilms can involve killing mechanisms such as those using antibiotics, bacteriocins or extracellular membrane vesicles, or strategies that compromise growth such as nutrient depletion or the inhibition of quorum sensing.

cystic fibrosis

excess mucus production in the airways hosts bacteria such as Pseudomonas aeruginosa, which use dead leukocytes from the immune system to construct their biofilm coating

Bacterial EPS

extracellular polymeric substance or extracellular polysaccharide exopolysaccharide or exo-polymeric substance

mature biofilms contain differentiated cell populations, including:

flagellated, and matrix producing populations

biofilm growth

form a film by working together (pellicle formation)

Myxobacteria

form a fruiting body once nutrients run out. Some of the cells become dormant spores called "myxospores" these spores get transported to a different location via wind or rain to form a new colony in the new location. This group of organisms uses gliding motility. they lack a flagella. gliding motility only works if the bacteria is in contact with a surface.

planktonic growth

free floating suspension of cells not working together

what percent of all bacteria are found in biofilm

greater than 98%

what is a nosocomial infection?

hospital acquired infection

lotic environment

human mouth, stream of water, etc. biofilms on your teeth are in a lotic environment

what creates an ideal conditioning film for S. aureus attachment

human plasma

how can flagella help mediate biofilm formation?

if they bind to a surface, they can help mediate formation of a biofilm. very important in initial colonization of surfaces

Diffusion in biofilms vs planktonic culture

in biofilms: diffusion is much more critical, especially when it thickens. without diffusion, you get very different environments in the layers of the biofilm in planktonic cultures: diffusion is usually of little consequence

what happens in step 2 of biofilm formation?

irreversible attachment, and EPS secretion.

vibrio cholerae mutants

lacking attachment pili -doesn't go into orbital motion lacking c-di-GMP phosphodiesterase -hyper biofilm former

when did scientists and engineers possess adequate technology to effectively study the biofilm mode of growth?

late decades of the 20th century

what is the importance of pseudomonas?

model biofilm former

how much of the earth's biomass is biofilm?

more than 50%

How is EPS production important during infection?

mucoid strains of P. aeruginosa are commonly isolated from the cystic fibrosis lung because overproduction of the EPS alginate is beneficial for the bacteria during infection. This mucoid EPS is characteristic of aggregates where cells are good at adhering to each other.

what nutrient levels are thought to induce hyphae formation?

nutrient levels more closely resembling the human host environment

endocarditis

one of the four heart valves, the heart lining, or heart muscle is infected by bacteria (usually sreptococci). the formation of endocarditic plaque is unique, involving bacteria, platelets, coagulation factors and leukocytes

cyclic-di-GMP

signals to the cells that they should enter biofilm formation when accumulated at high levels.

Which class of molecule might be most successful at penetrating the typical biofilm?

small, uncharged

biofilms of Candida albicans are critical for what?

critical for virulence: -mutants unable to produce biofilms exhibit severely impaired infectivity

how are microorganisms adaptive?

depending on what genes they are expressing, they are able to adapt to different enviroments

sheer force can cause?

detachment

Step 5 in biofilm formation

detachment and dispersal -active or passive: active is if the microbe chooses to disperse, passive is if environment disperses it unwillingly

the presence of ________ is indicative of a truly mature biofilm

differentiated cell population

how do gradients form in biofilms?

diffusion is not 100%, low oxygen and low nutrient environments are observed deep in the biomass

passive dispersal mechanisms in biofilm infection

driven by the flow of blood

thought question: polysorbate 80 is a surfactant that inhibits biofilm formation. How does the surfactant qualities of this compound prohibit formation of biofilms?

eliminating the surface tension and hindering aggregation of cells.

what do biofilms impact in real world application

teeth, cooling water, oil recovery, food processing, drinking water, paper manufacturing, medical implants, ship hulls, drinking water

how are localized gradients important in biofilms?

the biofilm hinders diffusion of nutrients into the biofilm. the gradients create heterogeneity in the biofilm which creates different gene expression profiles in the biofilm

what happens in step 2 of biofilm formation?

the cells start to release some extracellular polymeric substance

how does B. subtilis decide which bet hedging strategy to use?

the decision is partially stochastic (or random) -there are mutually exclusive regulatory loops driving either competence or sporulation -you can either turn sporulation "ON" and competence "OFF" or vice versa -The opposing circuits can try to turn on at the same time, but since they are mutually exclusive only one or the other will randomly "win" -The decision is also partially a physiological adaptation to an environment.

biofilm maturation is shaped by?

the environment. ex) low Fe in environment produces a different biofilm as higher Fe available.

amyloid biofilm of S. aureus

the hydrophobic side digs its way into the membrane and form a pore. this is important if the cell was ingested by a phagocyte, it can lyse its way out.

Cell differentiation

the process by which a cell becomes more specialized, and expresses a set of genes that were not expressed in the undifferentiated cell while silencing other genes. traditionally thought of as a feature of multicellular organisms

what is microbial leaching?

the process of extracting metal from ores using microorganisms, can purify metals such as copper lead zinc gold silver and nickel with lower production costs

what is bioremediation?

the use of living organisms, such as prokaryotes, fungi, or plants, to detoxify a polluted area by degrading soil bound contaminates

EPS that are not covalently attached to the bacterial cell surface.

these are very important to infection, so if you delete the EPS gene from this strain the strain becomes avirulent

what is SasG?

this is a surface protein in staph aureus that allows attachment to each other

Antibiotic resistance

this is an actual genotypic change where you acquire genes that allow you to survive an antibiotic treatment. This can occur in polymicrobial biofilms where transduction transfers its resistance from one kind of cells to cells that do not have this resistance gene. This is more permanent.

antibiotic tolerance

this is more of the phenotypic changes. This is a change in the gene expression profile level. So they are capable of entering dormancy instead of being active and so in the dormancy they aren't susceptible to the antibiotic until they are out of dormancy. This is transient.

in the absence of a traditional conditioning film, how can S. aureus produce its own conditioning film?

through autolysis of a subpopulation of cells. The intracellular contents (including DNA) are released into the surroundings. The extracellular DNA (eDNA) provides a scaffold for surface attachment.

what is the purpose of forming a fruiting body?

to generate myxospores

true or false: iron starvation in biofilms in infection is well defined

true

how much more resistant to antibiotics are cells within a biofilm, as opposed to their planktonic counterparts?

up to 1000x

how are urinary catheters affected by biofilms?

urinary catheters are known to get clogged up by biofilms

FnBP-mediated biofilms

very characteristic of methicillin resistant S. aureus isolates

fibrin biofilm is very important during_____

very important during infection, (coagulase mediated)

nutrient scavenging properties of EPS

water channels form in the biofilm to help feed the biofilm

what is biological bet hedging?

when an organism suffers decreased fitness under "normal" conditions in exchange for increased fitness in stressful conditions.

what is a problem with chronic inflammation?

when our body is inducing inflammation, it is also killing our tissue. this leads to a lot of dead or damaged tissues which is good for biofilm formation.

In the case of a disease that is not associated with the biofilm form of a microbe, would it still be of interest to study the biofilm-forming ability of microbe in other contexts? why or why not?

yes

what type of biofilm morphology would you see in infection and why?

you would probably see the low iron morphology (flat, not cloud like) because following infection your body sequesters nutrients from the invader to try to prevent growth.

Watnick and Kolter described biofilms as __________

"microbial cities" with a complex mixture of ethnic neighborhoods, transportation and communication networks, mechanisms which protect the inhabitants from harm and constant urban renewal in which removal of some neighborhoods is balanced by the growth of new ones.

the importance of surface associated bacterial communities

"surfaces enable bacteria to develop in substrates otherwise too dilute for growth. Development takes place either as bacterial slime or colonial growth attached to surfaces"

Candida albicans

-dimorphic fungus that grows as both yeast (ovoid cells) and hyphae (filamentous cells) -most common human fungal pathogen -model organism for infectious biofilms

why is diffusion limited in biofilms?

-due to protective EPS -due to cell density

Antibiotic resistance

-genotypic changes (acquisition of genes) -polymicrobial biofilms increase genetic transfers between species

What are the negative influences of biofilms on human health and industry?

-Biofilms are notorious for causing pipe plugging, corrosion and water contamination -Biofilms are responsible for billions of dollars in lost industrial productivity, as well as product and capital equipment damage each year

What are some ways we can manipulate biofilms to our benefit?

-Bioremediation: biofilms attached to particles of contaminated soils and aquatic sediments can help degrade soil-bound contaminates that occur from accidental chemical releases into the environment -Microbial leaching: the process of extracting metal from ores using microorganisms, can purify metals such as copper lead zinc gold silver and nickel with lower production costs

Polysaccharide biofilm of S. aureus

-dependent on expression of poly N-acetylglucosamine (PNAG) AKA polysaccharide intercellular adhesion (PIA) -common in methicillin-sensitive s. aureus isolates -produced by intracellular adhesion operon

What are the fundamental differences between biofilm and planktonic cells?

-different phenotypes -biofilms have differentiated cells -planktonic cells can diffuse easily

what are the microbial benefits to biofilm formation?

-ability to survive in nutrient deficient conditions -protection from antibiotics, disinfectants, and other environmental stressors

Roles of cell types- Hyphae

-can physically penetrate barriers: promote tissue destruction and invasion -resistant to immune cells: the filamentous shape is more difficult for phagocytes to engulf

Fibrin biofilm of S. aureus

-coagulase-mediated conversion of fibrinogen (Fg) into fibrin -Fibrin is formed as a part of normal blood clotting -fibrin can be recruited into a biofilm scaffold -coagulase positive strains dominate the known pathogens of staph genus.

which of these is the BEST term describing EPS? A) extracellular polymeric substance B) extracellular polysaccharide C) exopolysaccharide D) exo-polymeric substance E) none of these

A) extracellular polymeric substance

EPS of P. aeruginosa

3 primary types 1) Psl 2) Pel 3) alginate

ecosystem

A biological community of interacting organisms and their physical environment.

what is a protein/gene that induces active detachment and dispersal

Alginate lyase protein (gene: algL) is an enzyme that catalyzes the depolymerization of alginate. It also is required for alginate export.

what microorgansim has multiple bet hedging strategies to ensure survival?

B. subtilis

S. aureus surface proteins that mediate direct cell to cell contact during protein/eDNA biofilm accumulation

BAP, FnBP's, SasG, Aap (Biofilm associated protein) (Fibronectin-binding proteins) (S. aureus surface protein) (Accumulation-associated protein)

capsules on what bacteria are critical to infection

Bacillus anthracis Streptococcus pneumoniae -if these two are noncapsulated, they become avirulent.

capsules critical to infection

Bacillus anthracis (anthrax) S. pneumoniae

What are the ways in which we can use these biofilm associated mechanisms to our benefit?

Beneficial biofilms include: -roles in oxygen production -basis for food webs that nourish larger organisms -recycling of elements -most plant associated microorganisms assist with plant's ability to absorb nutrients from the soil -most human associated microorganisms: non-pathogenic gut and skin microbiome assists in the protection from pathogens

which S. arueus biofilm type is least likely to occur in a laboratory test tube? A) Polysaccharide B)Protein/eDNA C)Fibrin D)Amyloid

C) Fibrin fibrin is host-derived and therefore would not be found in a standard laboratory test tube

which of these is not typically a requirement for a good model organism A) genetically tractable B) easily grown C) pathogenic D) displays processes evolutionarily in other organisms C) all of these

C)pathogenic

B. subtilis bet hedging strategies in times of stress: Competence

Competence: the ability of a cell to take up DNA from the environment. -Risks: you are more likely expending energy to take in useless DNA or even damaging DNA deriving from a virus. -Advantages: you might acquire a useful gene that can get you through the stress condition. ex) antibiotic resistance gene

what molecules are absorbed by the biofilm

Depending on the composition of the EPS. -generally small, uncharged particles are absorbed -positively charged molecules can be absorbed due to the negatively charged EPS

common causes of UTI

E. coli (63%)

B. subtilis bet hedging strategies in times of stress: Endospore formation

Endospore formation: highly differentiated cells resistant to heat, harsh chemicals, and radiation. -present only in some gram positive bacteria. other organisms have similar types of dormancies that just aren't quite as resistant as endospores. (ex. persister cells) -dormant stage of bacterial life cycle. -vegetative cells are metabolically active and capable of growth, endospores are not.

fimbriae of E. coli virulence factors

Fimbriae: -composed of specific bacterial proteins -more numerous and usually shorter than flagella -facilitate adherence, not motility

Myxococcus xanthus

Fruiting body development: -type of myxobacteria -myxococcus xanthus uses gliding motility (non flagellated) -starvation triggers the aggregation of 100,000 cells which form a fruiting body -forms myxospores

what is the EPS important for?

In Irreversible attachment step, EPS is important for protection, for feeding cells, communication, and is full of plasmids that help with horizontal gene transfer.

Step 3 in biofilm formation

Microcolony formation -irreversibly attached cells begin to divide -clonal population originating from a single cell -aggregate of microbial cells containing around 50 cells or less

Biofilms of Candida albicans

Mixture of cell types -yeast -hyphae -pseudohyphae Critical for virulence -mutants unable to produce biofilms exhibit severely impaired infectivity

fruiting body development

Myxococcus xanthus uses gliding motility (non-flagellated) -starvation triggers the aggregation of 100,000 cells which form a fruiting body

what is a surface protein in S. aureus that allows cell to cell attachment?

SasG

B. subtilis bet hedging strategies in times of stress: Sporulation

Sporulation: formation of a tough and dormant non-reproductive structure (endospore) -Risks: you are no longer reproducing and evolving to fit the environment. If you exit dormancy too soon, you are dead. -Advantages: you are protected from the environment as long as you stay in the spore

Common causes of acute otitis media

Streptococcus pneumoniae Haemophilus influenza Moraxella catarrhalis Staphylococcus aureus

why do pathogens usually form biofilm communities during infection?

The human body is an ideal surface, mechanisms of biofilm recalcitrance and persistance in human infection -antibiotic tolerance -using diffusion reaction inhibition -the antiimmune system (limited penetration of phagocytes, decreased phagocytic capacity)

what is polysorbate 80?

This is a surfactant that inhibits biofilm formation in P. aeruginosa.

vibrio cholerae motility that promotes attachment

orbital motility promotes attachment to surface

coagulase positive strains species tend to be?

pathogens (most strains of S. aureus)

Biofilms have a distinct __________ compared to planktonic cells

phenotype

what are the two distinct modes of growth?

planktonic biofilm

what are the four types of biofilms that S. aureus forms and what are they characterized by?

polysaccharide biofilm protein/eDNA biofilm fibrin biofilm amyloid biofilm these are all characterized by different EPS composition

extracellular matrix composition

polysaccharides proteins DNA lipids

Biofilms on your teeth are an example of

primary biofilms

Pseudomonas aeruginosa

produces both Pili (fimbriae) and flagellae which bind to different components of the host environment. They can colonize different surfaces on the epithelial cells due to their different surface proteins they are expressing

Biofilm active dispersal is mediated by?

proteases, alginate lyase, matrix degrading enzymes,

how do biofilms coordinate their behavior? (how do they communicate)

quorum sensing mechanisms: mode of bacterial communication