Lecture 4 - Biofilms

Question 1

Biofilm is defined as a community of _____ attached to a ______ (biotic or abiotic), encased in a ________.

Most biofilms are __________.

_____ bacteria can form a biofilm under certain conditions.

Answer 1

microbes; surface; matrix.

multispecies

most

Question 2

What are planktonic cells?

Answer 2

Free-swimming cells.

These cells can make biofilms when gathered as a community.

Question 3

(T/F) Biofilms were observed in the lab first.

Answer 3

False.

Biofilms were observed in nature (freshwater) before the lab.

“it is quite evident that for the most part the water bacteria are not free floating organisms, but grow upon submerged surfaces”

Question 4

Briefly describe the biofilm development cycle.

Answer 4

Planktonic cells utilize motility, adhesins, and environmental cues, followed by quorum sensing, to develop into mature biofilms. Some cells detach from the biofilm and return to a planktonic state.

Detached cells can remain planktonic or start a biofilm somewhere else.

Question 5

Biofilms are ____-like structures.

These structures have ______ for liquids to flow through allowing for _________ and _______.

Answer 5

pillar

channels; nutrient intake; waste removal

Question 6

Why form a biofilm?

Answer 6

1) slow growth = less energy output

2) nutrients are associated with surfaces (thus cells are on surfaces)!!

3) cooperation within community (easier in this env if one bacteria can make something required for another bacteria)

4) provides protection from antimicrobial agents, predators and the immune system

Question 7

Where are biofilms formed?

Answer 7

1) in the environment
2) in industry
3) in medicine

*basically everywhere that has some sort of nutrient and moisture

Question 8

Biofilms can grow on:

1) on medical equipments

2) on our organs!

3) in nature

Give distinct examples of each.

Answer 8

1) contact lens CASE and CATHETERS used for urine passage can be blocked by bacterial cells and matrix and lead to bad infections

2) S. aureus on CARDIAC tissue. P. aeruginosa in SPUTUM from a cystic fibrosis (CF) patient (example of biofilm not forming on real surface). - DENTAL PLAQUE (cause inflammation in gums –> heart problems)

3) Morning glory pool in yellowstone park (microbes colonize thermal pools and springs in the park & give them their distinctive and unusual colours).

Question 9

1) When started studying biofilms at a molecular level in the late 1990s, what was the hypothesis they were testing?

2) What were the two approaches used to address this hypothesis?

Answer 9

Hypothesis: surface-attached cells (biofilm) express different genes compared to planktonic cells.

Approaches to address this hypothesis:
1) DNA microarray analysis
2) 2D gel protein analysis

Question 10

A paper compared gene expression patterns of planktonic and biofilm cells using a P. aeruginosa DNA array.

1) How many genes did this array contain?

2) What did they find?

Answer 10

1) The array contained 5,500 of the predicted 5,570 genes.

2) 0.5% of the genes were activated in biofilms while 0.5% were repressed.

Activated genes included ones involved in protein translation and certain types of metabolism.

Repressed genes included ones involved in flagellar motility and secretion

Question 11

Briefly describe how 2D gel electrophoresis occurs.

Answer 11

First, there is separation of proteins in first dimension (by charge).

This gel is applied to the top of the second gel.

Then, there is a separation in second dimension (by size).

Question 12

A paper compared planktonic and biofilm cells expressed proteins using 2D gel analysis in P. putida.

What did they find?

Answer 12

P. putida biofilms major differences:

There are 15 up-regulated genes (pilis are up-regulated).

There are 30 down regulated genes (flagella is down-regulated).

Question 13

Several DNA microarray and protein expression studies have been published.

What discoveries have these studies revealed thus far?

Answer 13

Planktonic cells and cells growing in biofilms do express different set of genes but there are NO GENES IDENTIFIED THAT ARE ONLY EXPRESSED IN BIOFILMS.

No biofilm markers have been identified!

This may be due to the use of different strains and growth conditions in these papers.

Question 14

which statement is false?

1) most bacteria will form biofilms under certain conditions.

2) molecular aspects of biofilm formation tend to be studied for all bacteria.

Answer 14

2!

molecular aspects of biofilm formation tend to be studied for MEDICALLY or INDUSTRIALLY important bacteria.

Question 15

Briefly answer the following questions of P. aeruginosa:

1) Describe the bacterial structure and strain.

2) What kind of pathogen is it?

3) What are the advantages of studying with this bacteria?

Answer 15

1) Gram negative, motile rod

2) Opportunistic pathogen - pathogen of immunocompromised and burn patients as well as people with cystic fibrosis (CF)

3) sequenced genome, genetically amenable, and best studied model for biofilms

Question 16

Describe the steps of biofilm formation by P. aeruginosa

Answer 16

Planktonic cells initially attach to the surface in a reversible manner before transitioning into an irreversible attachment phase, leading to the FORMATION OF A MONOLAYER.

Then, there is the formation of a MICROCOLONY and then a MACROCOLONY (biofilm).

Question 17

What kind of assay did O’Toole and Kolter use to determine which genes are important for biofilm formation?

What was their hypothesis?

Answer 17

O’Toole and Kolter used 96 well microtitre plate as assay system.

They used P. aeruginosa random transposon insertion mutant library and screened 2400 mutants. They identified mutants that did not form biofilms and determined which genes were affected in the mutants.

Hypothesis: specific genes control biofilm formation.

Question 18

How did O’Toole and Kolter construct their random transposon-insertion mutant library?

Answer 18

They constructed their library by adding a transposon (tn) to wild type culture of P. aeruginosa.

Under specific conditions, transposon inserts RANDOMLY and only ONE TIME, into the chromosome of P. aeruginosa.

Among thousands of transposon-insertion mutants, should have an insertion in each non-essential gene (genes not required for survival).

If there is no biofilm, they can check which gene is mutated and know that it is important for biofilm.

Question 19

Describe the 96-well Microtitre Plate Assay.

Answer 19

The 96-well microtitre plate assay involves adding cells to the wells, allowing them to adhere and form biofilms over a period of time (in this case, 10 hours).

After incubation, the wells are rinsed to remove non-adherent cells, and then a dye such as crystal violet is added to stain the remaining bacteria in the biofilm.

The absorbance at 600 nm (A600) is then measured at different time points to monitor the development of biofilms over time.

Question 20

What mutants in O’Toole and Kolter paper did not form biofilms?

Answer 20

pilY1
pilB
flgK

Question 21

What is the advantages and disadvantages associated with 96-well Microtitre Plate Assay?

Answer 21

Advantage: high throughput
Disadvantage: gross level (yes or no) - can not tell if little bacteria stuck on the walls

Question 22

What is the flow cell system? How does it differ from microtitre plate assay?

Answer 22

Flow cell system is a flowing system where we can watch biofilm formation over time under the microscope.

There is a RESERVOIR with media that flows through the system. There is also a FLOW CELL with 4 channels/ grooves. Each channel is inoculated with different strains of bacteria at 37˚C. if bacteria is not attached, they will disappear with the flow.

A glass slip on top encloses channels; and this can be put on microscope and image bacteria.

It is less high throughput compared to the 96 well microtitre plate assay but it gives more information (less gross level).

Question 23

Briefly describe what you would see under a microscope of wildtype biofilm formation in a flow cell over 7.5 hours.

Answer 23

Within 30 minutes, there is initial attachment.

In 1.5 hours, there is formation of a monolayer.

In 5 hours, cells start to come together forming microcolonies and in 7.5 hours, microcolonies become macrocolonies.

Question 24

(T/F) flgK and pilB are important for biofilm formation at different stages.

Answer 24

True!

flgK mutants can’t attach.

pilB mutants can attach but are arrested at monolayer formation and can’t progress further.

Question 25

What does flgK encode for?

Answer 25

flgK encodes a protein that is part of the FLAGELLUM. Flagella is an extracellular structure important for swimming motility and/or ATTACHMENT TO SURFACES. Thus flgK mutants can't attach to form a biofilm!

Question 26

What does pilB encode for?

Answer 26

pilB encodes a protein important for the synthesis of TYPE IV PILI. Pili are short, thin proteinaceous fibers that cover entire surface of the cell. They are required for TWITCHING MOTILITY (used for testing where things are good to stick). *PilB mutants can form a monolayer but can't do twitching motility which is important for bacteria to come together thus can't move forward to mature biofilm.

Question 27

1) What kind of bacteria is E.coli? 2) What does it cause? 3) Do all strains of E.coli have the same virulence factors? 4) What is UPEC?

Answer 27

1) Gram negative bacteria 2) Causes diarrhea, dysentery, kidney failure, bladder infections and pneumonia 3) Virulence factors VARY with strain (one that causes diarrhea will not cause a UTI). Most strains are NON-pathogenic (live in our GI tract) 4) Uropathogenic E. coli (UPEC) causes UTI

Question 28

(T/F) E. coli biofilm formation differs from P. aeruginosa.

Answer 28

False! They follow the same steps: initial attachment -> monolayer formation -> microcolony formation -> macrocolony formation

Question 29

In E. coli biofilm formation, what is required in the transition from 1) planktonic to initial attachment 2) initial attachment to monolayer formation 3) monolayer formation to microcolony formation 4) microcolony formation to macrocolony formation

Answer 29

1) flagella 2) Type I pili / Ag43 3) flagella 4) colanic acid *colanic acid mutants form a microcolony but not macrocolony

Question 30

What are the (3) properties of a mature biofilm?

Answer 30

1) Surrounded by EXOPOLYSACCHARIDE MATRIX. 2) HETEROGENEITY-BACTERIA throughout the biofilm experience different environments. 3) Resistant to antimicrobial agents as a whole.

Question 31

1) What are the two main functions of the matrix? 2) How do we know this?

Answer 31

1) Structure and protection 2) Observation (microscopy) and identification of important elements of the matrix & then deletion of important genes to determine what their individual functions are

Question 32

What is the matrix composed of?

Answer 32

1) polysaacharides (sugars) 2) DNA (eDNA secreted into matrix by lysed cells) 3) proteins (not much known about these)

Question 33

What are the 3 sugars in the P. aeruginosa biofilm matrix?

Answer 33

1) Alginate 2) Psl (polysaccharide synthesis locus) 3) Pel *different bacteria = different sugars

Question 34

Match the sugars of the P. aeruginosa biofilm matrix to their definitions: 1) Alginate 2) Psl (polysaccharide synthesis locus) 3) Pel A) involved in pellicle formation (floating biofilm on liquid). glucose-rich polysaccharide B) overproduction occurs in P. aeruginosa strains isolated from CF patients. strains unable to produce this sugar still form similar biofilms. C) mannose and galactose rich polysaccharide

Answer 34

Alginate: overproduction occurs in P. aeruginosa strains isolated from CF patients. strains unable to produce this sugar still form similar biofilms. Psl: mannose and galactose rich polysaccharide Pel: involved in pellicle formation (floating biofilm on liquid). glucose-rich polysaccharide

Question 35

What is eDNA and its function?

Answer 35

eDNA is secreted by cells in the biofilm as an active process. Function: structure and protection of the cells in the biofilm

Question 36

Do bacteria in the biofilm experience the same environments? Give examples.

Answer 36

Heterogeneity bacteria throughout the biofilm experience different environments! - waste (more waste deep in the biofilm; outside can diffuse out) - nutrients (more nutrients on the outer bacteria; less inside the biofilm) - signalling molecules (gradient) - antibiotics (come from outside so outside cells are more susceptible than cells deep inside)

Question 37

Cells experiencing different environments in biofilms express _________ sets of genes.

Answer 37

different *heterogeneity bacteria throughout the biofilm experience different environments.

Question 38

What are some planktonic antibiotic resistance mechanisms?

Answer 38

Intrinsic (part of the genome) - low outer membrane permeability and action of efflux pumps Acquired (through mutation) - upregulation of drug efflux pumps and alteration of antibiotic target

Question 39

(T/F) Biofilm specific antibiotic resistance mechanisms are different than planktonic antibiotic resistance mechanisms.

Answer 39

True!

Question 40

For biofilm specific resistance mechanisms: 1) There are ______ mechanisms. 2) They are ________. 3) One example is through their ______. 4) These genes are ______ expressed in biofilms.

Answer 40

1) multiple 2) INTRINSIC (not based on mutations) 3) matrix (eDNA) 4) more

Question 41

Where (4 locations) can we find beneficial biofilms?

Answer 41

water treatment energy production (microbial fuel cells) nitrogen fixation biocontrol (protecting crops)

Question 42

1) what are the different stages of wastewater treatment? 2) what do biofilms do?

Answer 42

1) primary, secondary, and tertiary stages 2) biofilms (along with planktonic bacteria) in AERATION TANKS (2˚ stage) break down ORGANIC MATTER

Question 43

What are microbial fuel cells (MFCs)? What do they consist of?

Answer 43

Harness the energy potential generated when bacteria metabolize substrates (eg glucose). Consist of an ANODE, a CATHODE, a PROTON or CATION exchange membrane and an ELECTRICAL CIRCUIT

Question 44

Fill in the blanks regarding the MFC: Bacteria at _____ convert glucose to _____, ______ and ________. Oxygen is usually final electron acceptor but oxygen is not present here so the MFC electron acceptor is an _______. Then, the electrons flow through an electrical ______ to the _______. _______ ______ (watt) is generated due to the ________ difference (volt) between the anode and the cathode, along with the flow of ____ (ampere). At the _______, the electron acceptor is reduced (O2 -> H2O). Energy and H2O generated by bacteria!

Answer 44

anode; CO2; protons; electrons anode circuit; cathode electrical power; potential; electrons cathode

Question 45

(T/F) Half of MFCs is anaerobic (bacterium + anode) and the other half is aerobic (cathode).

Answer 45

True!

Question 46

(T/F) The use of MFCs is very economically competitive.

Answer 46

False! The use of MFCs is not yet economically competitive. The current focus is energy generation using products from wastewater treatment plants. Generation of energy and CLEAN WATER using products from any industrial process (such as beer pdt)

Question 47

1) which bacteria fix nitrogen? 2) which bacteria protect tomatoes from rot?

Answer 47

1) S. meliloti (alfalfa) and B. japonicum (soybean) fix nitrogen. 2) Pseudomonas fluorescens biofilms protect tomatoes from rot caused by fungus. example of BIOCONTROL (using a biological agent to control).

Question 48

Nitrogen is required by ____ living organisms. Earth's atmosphere is ____ nitrogen gas. This is unusable and must be fixed: _____ (combined with ______) to ammonia by nitrogen fixing bacteria. Green plants use the fixed nitrogen to produce ______ that are then passed onto the food chain.

Answer 48

all 80%; reduced; hydrogen proteins N2 + 3H2 --> 2NH3

Question 49

(T/F) The nodule of a clover contains S. meililoti where nitrogen fixation occurs.

Answer 49

True!

Question 50

Where can we find problematic biofilms?

Answer 50

1) industry (clogging & corroding pipes and marine biofouling) 2) environment (plant pathogens) 3) medicine *$30-60 million a year is spent due to an increased need for fuel because of increased drag - reduces speed and range of ship

Question 51

Which statement is true? 1) 20% of human bacterial infections are biofilm-based. 2) biofilms can form on anything you put in your body and they can form on tissue in your body. 3) biofilm-based infections marked by ongoing symptoms. 4) biofilm cells can be 2x more resistant to antibiotics compared to their planktonic counterparts

Answer 51

2! 1) 65% of human bacterial infections are biofilm-based. 3) biofilm-based infections marked by recurring symptoms after each bout of treatment with antibiotics. 4) biofilm cells can be 10-1000x more resistant to antibiotics compared to their planktonic counterparts!

Question 52

1) What are some implant infection examples of biofilms in medicine? 2) what are some diseases of biofilms in medicine?

Answer 52

1) catheters, artificial hips, and contact lenses 2) otitis media, periodontitis, recurrent UTIs, and cystic fibrosis (CF)

Question 53

Describe the mechanism of the RECURRING NATURE of biofilm based infections.

Answer 53

Biofilms can act as reservoirs for bacteria to hang out where they DO NOT EXPRESS virulence factors. Planktonic cells cause symptoms of acute infection by EXPRESSING virulence factors like toxins. Antibiotics kill the planktonic cells but not the biofilm cells. Biofilm cells can become planktonic cells through detachment signals. This is where the recurring nature of biofilm-based infections comes in.

Question 54

(T/F) While acute infections express virulence factors (planktonic cells), chronic infections do not express virulence factors (biofilm cells).

Answer 54

True!

Question 55

Give four examples of medically important biofilms. How does research approach medically important biofilms?

Answer 55

1) P. aeruginosa biofilms in CYSTIC FIBROSIS 2) E. coli biofilms in UTIs 3) H. influenzae/S. pneumoniae biofilms in CHRONIC OTITIS MEDIA 4) Diabetic foot ulcers Research focuses first on providing EVIDENCE that an infection is BIOFILM-BASED then the focus becomes identifying approaches to ELIMINATE the biofilm.

Question 56

1) What is cystic fibrosis? 2) What do CF patients develop?

Answer 56

CF is the MOST COMMON fatal GENETIC disease to affect young canadians. It results from a mutation in the CFTR (cystic fibrosis transmembrane conductance regulator; a chloride pump). CF patients develop a CHRONIC (can last for decades) BACTERIAL INFECTION in their lungs that persists despite extensive antimicrobial therapy. Therapy can manage symptoms so that there are stable periods. Exacerbations are periods where symptoms worsen and patients require hospitalization.

Question 57

1) What is the role of P. aeruginosa in CF patients? 2) Where is the evidence from for biofilm in the CF lung?

Answer 57

1) P. aeruginosa can attach to lung tissue in Cystic Fibrosis patients. P. aeruginosa chronic infections are responsible for mortality in over 90% of all CF patients. 2) Based on quorum-sensing autoinducer profiles (indicative of biofilm growth as opposed to planktonic growth). Microscopy.

Question 58

What is P. aeruginosa doing in the CF lung? How does it do that?

Answer 58

P. aeruginosa wants to PERSIST (remain in the lung). Classic virulence factors (such as ones that cause overt damage) tend NOT TO BE EXPRESSED when cells are growing in a biofilm. One important P. aeruginosa virulence factor is the T3SS; not expressed in the CF lung (chronic) but is expressed during pneumonia (acute).

Question 58

How does P. aeruginosa cause damage in CF?

Answer 58

Immune cells are recruited to the site (of biofilm) but cannot clear the infection. In the process of trying to clear the infection, lung tissue is damaged (through proteases and free radicals produced by immune cells). This is how P. aeruginosa causes INDIRECT damage to the lung tissue. Thus, the primary virulence factor is biofilm formation. *the thick mucus layer promotes biofilm formation

Question 59

What is the CF lung environment?

Answer 59

thick mucus and low oxygen

Question 60

Because the CF lung environment is low oxygen, a hypothesis generated was that P. aeruginosa biofilms will behave differently when they are grown anaerobically (and maybe this will result in a novel approach for therapy). 1) What was discovered when P. aeruginosa biofilms were grown under aerobic and anaerobic conditions? 2) What was discovered in the 2D gel analysis of biofilms grown aerobically and anaerobically? 3) What is a therapeutic question generated from these experiments?

Answer 60

1) More biofilm growth in anaerobic conditions! 2) OprF (porin) protein is present in anaerobic biofilm but not aerobic. 3) Would inhibiting the expression or function of OprF help CF patients?

Question 61

Which statements are true? 1) Mutations accumulate over time as P. aeruginosa ADAPTS to the CF lung. Slow growth = stressful conditions = accumulation of mutations in bacteria. 2) Most CF patients become infected with P. aeruginosa by age 5 and remain infected for 20-30+ years. 3) CF patients are often given antibiotics prophylactically to prevent exacerbation = stressful conditions for bacteria.

Answer 61

1 & 3! 2) Most CF patients become infected with P. aeruginosa by age 10 and remain infected for 20-30+ years.

Question 62

Certain mutants in P. aeruginosa are selected for over time in the CF lung. What are they?

Answer 62

1) mucA mutations (results in MUCOIDY) 2) mexZ mutations (mexZ is a negative regulator of MexXY which is an efflux pump that can increase resistance to aminoglycosides)

Question 63

Most strains of P. aeruginosa isolated from CF patients are _____ (not going in, but they become). This means there is an increased production of ________. This is generally due to mutation in _____ (negative regulator of _____ expression). _______ attenuates the inflammatory response (help PA avoid detection by immune system). Appearance of _____ strains correlates with _____ clinical outcome.

Answer 63

mucoid alginate mucA; alginate alginate mucoid; poor

Question 64

(T/F) P. aeruginosa is the only pathogen found in the CF lung.

Answer 64

False! P. aeruginosa is major pathogen but there are MANY OTHER bacteria, fungi and viruses that are typically in the CF lung. *Need to understand exact contribution of P. aeruginosa to CF (direct effects and indirect effects through interaction with other microbes and viruses)

Question 65

What are UTIs?

Answer 65

UTIs are considered to be one of the most common of bacterial infections. They tend to recur despite antibiotic therapy. Most common isolate is uropathogenic E. coli (UPEC).

Question 66

Because UTIs have a high incidence of recurrence, the hypothesis "biofilm are the cause of recurrent UTIs" was tested by Anderson. What was the evidence generated that proved this hypothesis to be true?

Answer 66

They looked at E. coli pods (E. coli attached to surface). They found pods on surface on infected mouse bladder with pathogenic E. coli, while there were no pods on mouse infected with non-pathogenic E. coli! The pods were present in a different type of mouse (a control). Z-series showed that bacteria are located throughout the pod. Further, E. coli pods present expressed factors important for biofilm formation (pili, Ag43, and polysaccharides).

Question 67

1) How do scientists approach the control of biofilm formation? 2) Since we know quorum sensing is a cell density sensing system that is important for many community-based behaviours, what was the hypothesis scientists wanted to test?

Answer 67

Identify genes important for biofilm formation and develop approach to prevent their function. For example, the O'Toole and Kolter screen for biofilm deficient mutants. Hypothesis: Quorum sensing (QS) is important for biofilm formation. This allows them to identify genes important for biofilm formation and develop approach to prevent their function.

Question 68

What are the two major QS systems in P. aeruginosa?

Answer 68

1) Las: lasI makes one type of autoinducer and lasR is the receptor that binds the autoinducer & affects gene expression. 2) Rhl: RhlI and RhlR are part of the second major QS system

Question 69

How did scientists discover that QS is involved in biofilm formation of P. aeruginosa?

Answer 69

They detected no macrocolony formation with a lasI mutant! No lasI = no autoinducer. When an autoinducer was added to the lasI mutant, the macrocolony was restored! Thus, a lasI mutation (QS factor) affects biofilm formation.

Question 70

Scientists were trying to discover if we can prevent biofilm formation by interfering with QS since biofilm formation is controlled by QS. They use a swarming motility assay to identify QS inhibitors? Why did they use it and what is it?

Answer 70

Motility is a QS controlled process. Thus, scientists used a swarming (community based motility) as an assay system to identify QS inhibitors. Cells are inoculated onto a centre of a plate. The cells grow and establish a colony. Motile swarm cells emerge at the edge of the colony. Colony spreads via swarming motility of the swarm cells and the growth of the inner cells. They tested to see if a suspected QS inhibitor affected the swarming motility (and thus QS).

Question 71

How was the quorum sensing inhibitor discovered? Describe the process of generating and testing this inhibitor.

Answer 71

Bacterial growth was not observed on this Australian seaweed (delisea pulchra) even where other seaweed host growth. Thus scientists hypothesized that D. pulchra produces an anti-biofilm compound. They isolated the QS inhibitor by FRACTIONATING (using different columns) the seaweed (D. pulchra). They performed initial experiments (using SWARMING assay or any other QS-based system) with CRUDE fractions which gave positive results. Then, they fractionated the crude fractions to isolate a QS inhibitor called a FURANONE.

Question 72

What was the affect of increasing concentrations of furanone 2 on swarming motility? What does this suggest?

Answer 72

Increasing concentrations of furanone 2 on swarming motility = decreased swarming with complete inhibition of swarming at high concentrations. Results suggests that furanone 2 disrupts quorum sensing!

Question 73

Scientists tested furanone (a compound that prevents quorum sensing) on preventing biofilm formation in the real world. What did they find?

Answer 73

They found that the compound prevents biofilm formation in the real world also!

Question 74

How do we study biofilms?

Answer 74

studying biofilms in vivo (patient samples, environmental biofilm samples) is difficult. most labs set up in VITRO monospecies biofilms. can vary growth medium to try to mimic in VIVO setting (eg synthetic CF sputum media). many labs trying to increase complexity of in vitro biofilms by introducing additional species (eg P. aeruginosa and E. coli)

Question 75

How do we detect biofilms?

Answer 75

Count the bacteria stuck to a surface (vortex, scrape, sonicate the cells off the surface and grow them up on nutrient plates). Visualize the bacteria (label them with something like a dye or a fluorescent marker and then look at them under a microscope)

Question 76

How do we get rid of biofilms?

Answer 76

1) Identify biofilm formation determinants and try to inhibit them 2) Identify factors that organisms use to INHIBIT other bacteria from forming biofilms 3) Identify factors that DECREASE RESISTANCE to antimicrobial agents 4) Identify DETACHMENT DETERMINANTS and try to express them 5) Identify organisms that PREY ON bacteria in biofilms

Question 77

Cells in biofilms can detach as single cells or in clumps as a natural part of the biofilm lifecycle. How did scientists identify a detachment factor?

Answer 77

Identification of a detachment factor: observation: P. aeruginosa detached from a flow-cell biofilm once the flow (no fresh nutrients) has stopped (several hours). hypothesis: some molecule builds up in the media that is a signal for detachment (this molecule should be present in SPENT - the media that the cells have been growing in for several hour). approach: add back cell-free spent medium (CSM) from P. aeruginosa cultures. they saw cells detaching from the biofilms, ultimately leading to no biofilm. they fractionated spent media and identified active fraction (cis-DA = cis-2-decenoic acid, a fatty acid)!

Question 78

What happened when cis-DA was added to biofilms?

Answer 78

When cis-DA was added to biofilm; the cells detached, ultimately leading to no biofilm in 30 mins. Similar results against E. coli, S. aureus and fungus were observed!

Question 79

________ are gram (-) predatory bacteria found in ___, ____ environments. They attack other gram (-) cells by ______ their periplasm, multiplying and ________ the cell envelope.

Answer 79

B. bacteriovorus wet; aerobic environments. entering; bursting *predatory bacterium reduces E. coli biofilm!