Biofilms and polymicrobial infections

Question 1

What is the definition of a classical infection?

Answer 1

Single organism of exogenous source (i.e. not part of normal flora).
Organism colonises susceptible host where it multiplies and evades host defence.
It damages host, normally by production of protein toxin (exotoxin)

Question 2

What is a polymicrobial infection?

Answer 2

Where numerous species/microbes are involved, no single organism associated with disease.
Impossible to apply Koch’s postulates to this scenario

Question 3

Outline how an initial viral infection in the lungs could lead to a secondary bacterial infection

Answer 3

1. Initial viral infection causes damage to lung tissue
2. Tissue damage then exposes basement membrane elements (e.g. fibrinogen) to which bacteria can adhere and infiltrate into the host
3. Viral neuraminidase cleaves sialic acid residues on host cells. This creates more bacterial binding sites.(neuraminidase inhibitors reduce s pneumoniae infiltration in experimental models)
4. Bronchitis or pneumonia cause by strep pneumoniae, haemophilus influenzae or S. Aureus
5. Impaired host immune response

Question 4

How could an over-activity of the immune system lead to infiltration of bacteria, lymphocytes, neutrophils and macrophages?

And what is the consequence of this?

Answer 4

Over production of inflammatory cytokines

(in the lung scenario): alveolar architecture is damaged.

Secondary bacterial infection leads to more prolonged and severe clinical symptoms when compared to viral infection alone. This bacterial invasion can lead to septicaemia - can lead to death.

Question 5

What 3 factors underpin septicaemia?

Answer 5

Virus
Bacteria
Host immune response

Question 6

What is the definition of a biofilm?

Answer 6

Biofilms are complex and dynamic structures.
A matrix-enclosed population of microbes that can adhere to biotic and abiotic substrates.

Biofilms are the most prevalent manifestation of polymicrobial communities.

Question 7

Give 3 examples of biotic substrates

Answer 7

Skin
Mucosal surfaces
Teeth

Question 8

Give 3 examples of abiotic substrates

Answer 8

Dentures/acrylics/resins
IV/urinary catheters
Abdominal drains
Stents
Ventilator tubes
Feeding tubes
Contact lenses
Heart valves

Question 9

Why does the composition of a biofilm change?

Answer 9

It changes in response to changes in the environment

Question 10

What 2 adjectives would you use to describe biofilm development in the oral cavity

Answer 10

Continuous

Sequential

Question 11

Name 2 examples of early colonisers in the dental biofilm

Answer 11

Strep sp.

A. Naeslundii

Question 12

Name 3 examples of late colonisers in the dental plaque biofilm

Answer 12

P. gingivalis
T. Denticola
T. Forsythia

Question 13

Which bacteria acts as a bridging species in the dental plaque biofilm?

Answer 13

F. nucleatum

Question 14

What does EPS stand for?

Answer 14

Extra polymeric substances

Question 15

What are mature biofilms encased in?

Answer 15

A matrix of extrapolymeric substances

Question 16

Name the 4 substances that are extrapolymeric substances (EPS)

Answer 16

Polysaccharides
Proteins
Lipids
Extracellular nucleic acid

Question 17

What are the 4 advantages of the EPS matrix?

Answer 17

1. Mechanical stability
2. Facilitates cell to cell interaction
3. Reduced efficacy of antimicrobials/immune cells
4. Microbes that grow in a biofilm are less sensitive to antibiotics than those that live free in suspension

Question 18

Outline the advantages of life in a biofilm over planktonic growth (5)

Answer 18

1. Increased metabolic fitness: nutritional cooperation
2. Increased genomic diversity: horizontal gene transfer antibiotic resistance
3. Increased stress resistance (biological/chemical/physical)
4. Aerobic bacteria can lower oxygen tension providing the means for anaerobic species to survive
5. Recalcitrance: reduced antibiotic penetrance into the biofilm, reduced antibiotic diffusion within the biofilm

Question 19

In what 2 states could you find an oral biofilm

Answer 19

healthy or dysbiotic

Question 20

What are the 3 basic steps of biofilm formation?

Answer 20

1. Adhesion
2. Microcolony
3. Mature biofilm

Question 21

What type of microbes are more abundant in a healthy biofilm?

Answer 21

gram positive facultatives

Question 22

What type of microbes become more abundant as a biofilm ages and develops towards disease?

Answer 22

Gram negative anaerobes

Question 23

Why is periodontitis a biofilm disease?

Answer 23

It is a polymicrobial infection of the subgingival crevice.

It is associated with a shift in gram status of the microbiota and chronic inflammation of the gums.

Question 24

Describe the transition in resident microbiota from healthy to peridontitis

Answer 24

Healthy microbiota > [Subtle changes in COMPOSITION of microbiota] GINGIVITIS > [Stabilisation of dysbiotic polymicrobial community] PERIDONTITIS

Question 25

What are the consequences of periodontitis (2)

Answer 25

Damage to structures supporting the tooth. | Resorption of alveolar bone - tooth loss.

Question 26

What are 3 examples of biofilm infections other than perio?

Answer 26

``` Medical plastics (e.g. catheters) Prosthetic heart valve endocarditis Contact lens keratitis ```

Question 27

Alongside high blood sugar, what triggers diabetic foot ulcers (DFU)?

Answer 27

Inappropriate foot care | Foot injuries

Question 28

What 3 underlying conditions could be involved in diabetic foot ulcers?

Answer 28

Nerve damage (neuropathy) Reduced blood flow (microangiopathy) Chronic inflammation

Question 29

What would the microbiota be like in a case of diabetic foot ulcers?

Answer 29

Host microbiota is dysbiotic | Biofilms formed

Question 30

How would you describe the polymicrobial interactions within biofilms?

Answer 30

Numerous, dynamic and complex

Question 31

Name the 3 polymicrobial interactions found within a biofilm

Answer 31

1. Physical interaction > co-aggregation 2. Chemical interactions > quorum sensing 3. Nutritional interaction > Digestive consortiums

Question 32

What is co-aggregation?

Answer 32

It is a process whereby genetically distinct bacteria attach to each other via specific molecules called adhesins

Question 33

What are planktonic cells?

Answer 33

They are free-swimming cells

Question 34

What can co-aggregates influence in a polymicrobial biofilm?

Answer 34

It can influence the development and composition of the biofilm

Question 35

Outline the interaction between Candida albicans (fungi) and S. Aureus (bacteria) Also give an example of where this interaction may be found in a real-life scenario

Answer 35

C Albicans produces hyphae, which are pathogenic invasive filaments. S. aureus can adhere to this hyphae. Systemic bacterial infection is facilitated by invasive hyphal filaments. Scenario: hospitals - catheters, IV, abdominal drains frequently affected

Question 36

Outline denture stomatitis and how it is a biofilm mediated condition

Answer 36

Polymicrobial biofilm-mediated condition Biofilm accumulates on dentures and is colonised by fungi Constant contact of fungal/bacterial biofilm with the oral mucosa Poor dental hygiene and failure to remove dentures allow bacteria, fungi and associated virulence factors to cause inflammation

Question 37

What is quorum sensing?

Answer 37

It is the regulation of gene expression in response to fluctuations in cell-population density.

Question 38

What do microbes secrete in quorum sensing?

Answer 38

Quorum sensing molecules (QSM)

Question 39

When quorum sensing molecules (QSM) are present in sufficient concentrations what happens?

Answer 39

They induce the expression or repression of quorum-dependent target genes. i.e. changes in transcriptional activity

Question 40

At what point in quorum signalling does specific changes in population behaviour occur?

Answer 40

When a critical threshold of signalling is exceeded

Question 41

Name 3 bacterial quorum sensing molecules

Answer 41

Autoinducer-2 (G negative and gram positive bacteria) N-Acyl Homoserine Lactones (NAHL) (Gram negative bacteria) Competence signalling peptides (Gram positive bacteria)

Question 42

Name a QSM that inhibits Candida Albicans filamentation

Answer 42

Farnesol

Question 43

What is particularly important for biofilm PERSISTENCE?

Answer 43

Acquisition of nutrients

Question 44

What are the principal substrates available in the mouth?

Answer 44

Proteins Sugars Glycoproteins (albumin, mucins, transferrins)

Question 45

If individual microbial species cannot break down substrates on their own, how do they gain nutrients?

Answer 45

Via digestive consortiums - species cooperate. Complex substrates are broken down into various sub sections by different species until they are simple molecules and can be utilised. At each step of the breakdown, a different molecule is made and can be utilised by a different species.

Question 46

What are accessory pathogens?

Answer 46

They are commensal microbes that can support or enhance the virulence of a different organism

Question 47

If endogenous oral microbes are introduced into a normal sterile tissue - what happens? Is it just one type of bacteria that causes this or is it polymicrobial?

Answer 47

Abscess Polymicrobial

Question 48

Name 3 ways microbial communities cooperate to avoid host immune responses

Answer 48

- Subversion of complement (P gingivalis, T forsythia) - Manipulation of neutrophils (P gingivalis) - Inhibition of macrophage responses (P Gingivalis)

Question 49

Roughly how many different bacterial species can be in a single periodontal pocket?

Answer 49

over 100

Question 50

How does some 'unculturable' bacteria i.e. bacteria that do not grow by themselves grow?

Answer 50

They have 'helper' strains

Question 51

What may be problematic if an infection is composed of multiple species?

Answer 51

Detection and Identification

Question 52

What are the issues with diagnosing polymicrobial infections?

Answer 52

It is time consuming and costly. Organisms are present in varying numbers - so sometimes difficult to detect lower levels of pathogens. Can be difficult to identify organisms to species level

Question 53

What technique can be used to identify the genus of microbes in a polymicrobial community?

Answer 53

16S rRNA next-generation sequencing and OTU determination

Question 54

Why is it difficult to treat polymicrobial infections?

Answer 54

- Very diverse: they display many complex phenotypes involving multiple virulence factors - Difficult to choose an individual antimicrobial; not all organisms may respond - Resistance to antimicrobials and antibiotics is growing concern - Outcomes are difficult to predict.