Biofilm and polymicrobial infections

Question 1

What is a classical infection?

Answer 1

One caused by a single organism.

The organism is of exogenous source (not part of normal flora).

The organism colonises susceptible host, multiples and evades host defence.

Causes damage to host, normally by production of protein toxin (exotoxin).

Question 2

What is a polymicrobial infection?

Answer 2

A number of micro-organisms at some sites of infection of multiple specifies. No single organism is associated with disease.

It can be a mixture of different species of the same type of microorganism or combinations of different types of organisms.

Kochs Postulates cannot be applied here.

Question 3

Give some oral examples of polymicrobial infection

Answer 3

Dental caries
Angular Cheiltitis
Dental Abcesses 
Dental Stomatitis 
Periodontal disease

Question 4

How does a secondary infection arise?

Give an example in lungs

Answer 4

Comes from a primary infection that then continues to get worse.

e.g. a viral infection followed by a bacterial infection

• Initial viral infection causes damage to lung tissue
• Tissue damage exposes basement membrane elements such as fibrinogen to which bacteria can adhere and infiltrate into the host
• Viral infection cleaves residues on host cell creating more bacterial binding sites.

Question 5

Give the overall mechanism of secondary infection

Answer 5

Over-activity of immune system

Overproduction of inflammatory cytokines leading to infiltration of bacteria, lymphocytes, neutrophils, macrophages.

Secondary bacterial infection leads to more prolonged and severe clinical symptoms when compared to viral infection alone.

Question 6

What is septicaemia caused by?

Answer 6

Initial viral infection, secondary bacterial infection and bad host response.

Question 7

How can we stop primary viral infections?

Answer 7

Do not use antibiotics as they do not work and can become resistant.

Can use antiviral and antibacterial therapy instead.

Question 8

What is the definition of the biofilm?

What is the definition of a biotic and abiotic substrate?

Answer 8

A matrix-enclosed population of microbes that can adhere to biotic and abiotic substrates.
(Biofilms are the most prevalent manifestation of polymicrobial communities.

They are complex and dynamic structures. The composition of a biofilm changes over time from person to person, between substrates and as a response to change in environment.

Biotic: skin/mucosal surfaces/teeth

Abiotic: dentures, acrylics, urinary catheters, heart valves, feeding tubes

Question 9

How does a biofilm develop?

Answer 9

Substratum coating.

Primary colonisers come and interact through adhesions onto acquired pellicle. They acquire nutrients, under cell division and form micro colonises.

Microcolonines produce EPS.
Secondary colonising species then come. Co-adhesion of cells occur .

Get transitions then. Diversity and complexity increases. Microbes within biofilm are subjected to selective pressure from external environment and within biolfim itself. Certain metabolites become depleted as the biofilm grows. Oxygen plays a role here. Waste material in biofilm increases due to metabolic reactions. This process is called succession - a pressure imposed on a developing population of microbes.

As a consequence of succession, the composition of the biofilm changes over time until equilibrium is achieved. This forms a mature biofilm, often called a climax community.

Question 10

What are some names of early colonisers?

Answer 10

Streptococcus sp
A nasetlundii
F nucleatum
P gingivalis

Question 11

Give some names of late colonisers

How do late colonisers interact with early ones?

Answer 11

T. forsythia

T. denticola

Can see these bacteria physically interact with each other through adhesions expressed on the surface.

Question 12

What is an extrapolymeric substance and what is it composed of?

What are the properties of the EPS?

Answer 12

EPS is a mature biofilm encased in a matrix of extrapolymeric substances.

• Polysaccharides
• Proteins
• Lipids
• Extracellular nucleic acid

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

Question 13

What are some advantages of life in a biofilm?

Answer 13

• Increased metabolic fitness: nutritional co-operation
• Increased genomic diversity: horizontal gene transfer and antibiotic resistance
• Increased stress resistance: biological/chemical/physical
• Aerobic bacteria can lower oxygen tension providing the means for anaerobic species to survive
• Recalcitrance: reduced antibiotic penetrance into the biofilm, reduced antibiotic penetrate within the biofilm

Question 14

What are the dynamics of biofilm maturation?

Answer 14

They can be healthy or dysbiotic.

Adhesion leads to a microcolony which leads to a mature biofilm. Increase in complexity of biofilm.

Health can turn to death. Shift of gram + to gram - disease.

Question 15

Explain how periodontitis is a biofilm disease

Answer 15

Polymicrobial infection of the subgingival crevice.

Associated with a shift in gram status.

Chronic inflammation of gums.

Damage to structures supporting the tooth.

Resorption of alveolar bone - tooth loss.

Transition in resident microbiota.

Question 16

How does a diabetic foot ulcer work?

Answer 16

Have high blood sugar.

Underlying conditions: nerve damage, reduced blood flow, chronic inflammation

Microbiota: dysbiotic host-microbiota, formation of biofilms

Triggers: inappropriate foot care, foot injuries

Question 17

What are the 3 main interactions of polymicrobes within biofilms?

Answer 17

1. Physical e.g. co-aggregation
2. Chemical e.g. quorum sensing
3. Nutritional e.g. digestive consortiums

Question 18

How does co-aggregation work?

Answer 18

Process whereby genetically distinct bacteria attach to each other via adhesins.

Co-aggregates are derived from planktonic cells.

Co-aggregates can influence the development and composition of polymicrobial biofilms.

Question 19

How do physical interactions in a biofilm work?

Answer 19

Candida albicans is a fungus that can produce pathogenic invasive filaments calls hyphae.

S.aureus can adhere to c.ablicans hyphae.

• Systemic bacterial infection is facilitated by invasive hyphae filaments
• Significant problem in hospitals where catheters and drains are frequently affected

Example: Denture Stomatitis - a polymicrobial biofilm condition. Biofilm accumulates on dentures and is colonised by fungi species.

• 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 20

What is quorum sensing?

Answer 20

The regulation of gene expression in repose to fluctuations in cell-population density.

Microbes secrete quorum sensing molecules. When present in sufficient conc, the molecules induce the expression or repression of quorum-dependant target genes- changes in transcriptional activity.

Specific changes in population behaviour occur when a critical threshold of signally is exceeded.

Question 21

What is a digestive consortium?

Answer 21

Where a variety of organisms change a complex substrate into a simple molecule.

e.g. glycoprotein to peptides to amino acids to short-chain fatty acid to a small molecule

can be done by removal of carbohydrate side chains, cleavage of protein, removal of terminal amino acids, methane production from fatty acids, reduction of sulphate

Question 22

Why do we get nutritional interactions in a biofilm?

Answer 22

Acquisition of nutrients is important for biofilm persistence.

Principal substrates avaliable in mouth include proteins, sugars and glycoproteins.

In general, individual microbial species are unable to completely degrade all of these substrates on their own.

Species can hover co-operate with each other to do so.

This is the digestive consortium.

Question 23

Explain the complexity of polymicrobial infections

Answer 23

Some disease-associated microbiota can be highly complex.

A single periodontal pocket can host over 100 different bacterial species.

Some species thrive in situ, but do not grow by themselves under standard laboratory conditions.

‘Unculturables’ are removed from infection site but unable to grow as a simple species without appropriate ‘helper’ strains. These require the addition of another microbe in order to survive.

If the infection is composed of multiple species, detection and identification can be problematic.

Question 24

How does polymicrobial co-operation and virulence work?

Answer 24

Microbes can persist as part of a polymicrobial community in harsh environments that would otherwise be lethal to them in mono-culture.

Accessory pathogens = commensal microbes that can support or enhance the virulence of a different organism

Introduction of endogenous oral microbes into a normally sterile site = abscess

Most oral bacteria are unable to form abscesses on their own in experimental abscesses models.

Minimum combination of 6-8 species required (polymicrobial co-operation)

Question 25

Is detecting polymicrobial infections easy or hard?

Answer 25

Detailed diagnosis is time-consuming and costly.

Organisms will be present in varying numbers. Sometimes difficult to detect those in smaller numbers e.g. keystone pathogens

Can be difficult identifying organisms to species level.

Question 26

How do we treat a polymicrobial infection?

Answer 26

The tremendous species diversity within polymicrobial communities presents a challenge for effective treatment.

These communities display a many complex phenotypes involving multiple virulence factors.

Resistance to anti-microbials and antibiotics is a concern.